High throughput assays for determining contractile function and devices for use therein

ABSTRACT

The present invention provides high throughput assays for identifying compounds that modulate a contractile function, as well as devices suitable for use in these assays.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/318,227, filed Oct. 31, 2011, which is a 35 U.S.C. §371 NationalStage filing of International Application No. PCT/US2010/033220, filedon Apr. 30, 2010, which claims the benefit of and priority to U.S.Provisional Patent Application Ser. No. 61/174,511, filed on May 1,2009. The entire contents of each of the foregoing incorporated hereinby reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grant numberN66001-08-C-2036 from the Defense Advanced Research Projects Agencyunder the United States Department of Defense. The United StatesGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Identification and evaluation of new therapeutic agents oridentification of suspect disease associated targets typically employanimal models which are expensive, time consuming, require skilledanimal-trained staff and utilize large numbers of animals. In vitroalternatives have relied on the use of conventional cell culture systemswhich are limited in that they do not allow the three-dimensionalinteractions that occur between cells and their surrounding tissue. Thisis a serious disadvantage as such interactions are well documented ashaving a significant influence on the growth and activity of cells invivo since in vivo cells divide and interconnect in the formation ofcomplex biological systems creating structure-function hierarchies thatrange from the nanometer to meter scales.

Efforts to build biosynthetic materials or engineered tissues thatrecapitulate these structure-function relationships often fail becauseof the inability to replicate the in vivo conditions that coax thisbehavior from ensembles of cells. For example, engineering a functionalmuscle tissue requires that the sarcomere and myofibrillogenesis becontrolled at the micron length scale, while cellular alignment andformation of the continuous tissue require organizational cues over themillimeter to centimeter length scale. Thus, to build a functionalbiosynthetic material, the biotic-abiotic interface must contain thechemical and mechanical properties that support multiscale coupling.

Accordingly, there is a need for improved methods and systems that maybe used for determining the effect of a test compound on biologicalrelevant parameters in order to enhance and speed-up the drug discoveryand development process.

SUMMARY OF THE INVENTION

Described herein are methods and devices for multiplex and highthroughput, high content measurements of physiologically relevantproperties of in vitro tissue constructs. The devices of the presentinvention can be used in, for example, high throughput screening assaysto determine the effects of a test compound on living tissue byexamining the effect of the test compound on various biologicalresponses, such as for example, cell viability, cell growth, migration,differentiation and maintenance of cell phenotype, electrophysiology,metabolic activity, muscle cell contraction, osmotic swelling,structural remodeling and tissue level pre-stress.

Accordingly, in one aspect, the present invention provides methods foridentifying a compound that modulates a contractile function. Themethods include providing a plurality of muscle thin films; contacting aplurality of the muscle thin films with a test compound; and determiningthe effect of the test compound on a contractile function in thepresence and absence of the test compound, wherein a modulation of thecontractile function in the presence of the test compound as compared tothe contractile function in the absence of the test compound indicatesthat the test compound modulates a contractile function, therebyidentifying a compound that modulates a contractile function.

In another aspect, the present invention provides methods foridentifying a compound useful for treating or preventing a muscledisease. The methods include providing a plurality of muscle thin films;contacting a plurality of the muscle thin films with a test compound;and determining the effect of the test compound on a contractilefunction in the presence and absence of the test compound, wherein amodulation of the contractile function in the presence of the testcompound as compared to the contractile function in the absence of thetest compound indicates that the test compound modulates a contractilefunction, thereby identifying a compound useful for treating orpreventing a muscle disease.

In one embodiment, the contractile function is a biomechanical activity,e.g., a biomechanical activity selected from the group consisting ofcontractility, cell stress, cell swelling, and rigidity.

In another embodiment, the contractile function is anelectrophysiological activity, e.g., a voltage parameter selected fromthe group consisting of action potential, action potential duration(APD), conduction velocity (CV), refractory period, wavelength,restitution, bradycardia, tachycardia, and reentrant arrhythmia; or acalcium flux parameter selected from the group consisting ofintracellular calcium transient, transient amplitude, rise time(contraction), decay time (relaxation), total area under the transient(force), restitution, focal and spontaneous calcium release.

In one embodiment, the methods of the invention further include applyinga stimulus to the plurality of muscle thin films.

In another embodiment, the plurality of muscle thin films are adhered toa solid support structure, e.g., a Petri dish, a multi-well plate, or aglass cover-slip.

In yet another embodiment, the plurality of muscle thin films arecultured in the presence of a fluorophore, such as a voltage-sensitivedye or an ion-sensitive dye. The voltage-sensitive dye may be anelectrochromic dye (e.g., a styryl dye and a merocyanine dye). Theion-sensitive dye may be a calcium sensitive dye (e.g., X-Rhod,aequorin, Fluo3, Fluo5, or Rhod2). In other embodiments, the fluorophoremay be a dye pair selected from the group consisting of di-2-ANEPEQ andcalcium green; di-4-ANEPPS and Indo-1; di-4-ANEPPS and Fluo-4; RH237 andRhod2; and, RH-237 and Fluo-3/4.

In some embodiments, the plurality of muscle thin films comprisescardiomyocytes, vascular smooth muscle cells, smooth muscle cells orskeletal striated muscle cells.

In another aspect, the present invention provides a device for measuringa contractile function. The device includes a solid support structure(e.g., a Petri dish, a multi-well plate, or a glass cover-slip); aplurality of muscle thin films adhered to the solid support structure,wherein the plurality of muscle thin films each comprise a flexiblepolymer layer (e.g., a flexible polymer layer comprisingpolydimethylsiloxane) and a population of isolated cells (e.g., myocytessuch as cardiomyocytes) seeded on the flexible polymer layer in apredetermined pattern, and wherein the cells form a tissue structurewhich can perform a contractile function. The plurality of muscle thinfilms may be adhered to the solid support structure directly orindirectly, e.g., via a plurality of posts attached to the solid supportstructure.

In a further aspect, the present invention provides a device formeasuring a contractile function which includes a solid supportstructure (e.g., a Petri dish, a multi-well plate, or a glasscover-slip); a plurality of muscle thin films adhered at one end to thesolid support structure, wherein the plurality of muscle thin filmscomprise a flexible polymer layer (e.g., a flexible polymer layercomprising polydimethylsiloxane) and a population of isolated cells(e.g., myocytes such as cardiomyocytes) seeded on the flexible polymerlayer in a predetermined pattern, wherein the cells form a tissuestructure which can perform a contractile function. The myocytes may bealigned to produce an anisotropic tissue.

In one embodiment, the devices of the invention may comprise a secondsolid support structure seeded with a second population of cells.

In another embodiment, the flexible polymer layer is supplied with anengineered surface chemistry before the cells are seeded. The engineeredsurface chemistry may include an extracellular matrix protein. In otherembodiments, the engineered surface chemistry is provided in a patternthat includes gaps.

In a further embodiment, the solid support structure includes aplurality of cell culture wells; an optical signal capture device; andan image processing software to calculate change in an optical signal.The optical signal capture device may further include fiber optic cablesin contact with the culture wells.

In another aspect, the present invention provides methods of preparing adevice suitable for measuring a contractile function. The methodsinclude providing a solid support structure; coating a sacrificialpolymer layer on the solid support structure; coating a flexible polymerlayer that is more flexible than the support structure on thesacrificial polymer layer, wherein the flexible polymer layer does notcover the edges of the solid support structure; seeding cells on theflexible polymer layer; culturing the cells to form a tissue; andremoving a portion of the formed tissue thereby generating strips of theformed tissue adhered at one end to the solid support structure, therebypreparing a device suitable for measuring a contractile function.

The present invention is further illustrated by the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a device of the invention in which a homogeneous cellpopulation is used. (A, B) Schematic representations of the device, withthin polymer films adhered to posts. (C) Photograph of the same device.

FIG. 2 depicts the system of FIG. 1 that further includes a co-cultureslide. (A, A′) depicts the embodiment wherein a co-culture slide iscultured separate from the device and (B, B′) inserted into the deviceprior to performing the methods of the invention. (C, C′) depicts theembodiment wherein the thin films are adhered to posts parallel to thedish base for optimal viewing.

FIG. 3 provides illustrations of a thin film adhered to a post. (A)depicts the embodiment wherein the polymer side of thin film adheres topost. (A′) depicts the embodiment wherein a PDMS thin film adheres to aPTFE post through hydrophobic-hydrophonic interaction. (B,B′) depictsthe embodiment wherein a change in the radius of curvature (R) can beused to calculate a change in stress in a cell layer.

FIG. 4 provides images and charts of the system of FIG. 1 usingrectangular-shaped muscular thin films with vascular smooth muscleanisotropically aligned along its length, and the deformation of themuscular thin films with time after treatment with endothelin-1. (A)depicts stereo microscopic images of polymeric thin films, seeded withvascular smooth muscle, adhered to posts. (B) depicts threshold imagesof thin films with films isolated from background. (C) depicts themeasured change in radius of curvature with time. (D) depicts musclestress necessary to induce change in curvature.

FIG. 5 schematically depicts the fabrication and use of a multi-wellbased device of the invention.

FIG. 6 graphically depicts two approaches for imaging the MTFs in thehigh-throughput methods described herein. FIG. 6(A) depicts imaging andmapping of all the wells of a multi-well plate assay at once and FIG.6(B) depicts imaging and mapping of each well of a multi-well plateassay individually.

FIG. 7A schematically depicts one embodiment of the fabrication and useof a horizontal MTF device of the present invention. FIG. 7B is aphotograph and image processing of the same device. FIGS. 7C and 7Ddepict the calculation of the radius of curvature of the MTFs in thedevice depicted in FIG. 7A.

FIG. 8 schematically depicts the contrast between assaying contractilityof an MTF using a large, rectangular glass comprising a horizontal MTFversus a round cover-slip comprising a horizontal MTF. (A) Fabication ofa horizontal MTF using a round cover-slip; (B) Fabication of ahorizontal MTF using a rectangular cover-slip (higher througput); (C)Contrast of running a contractility assay with a round cover-slip versusa square cover-slip. Although the manual fabrication of horizontal MTFis more efficient using square cover-slips, round cover-slips are oftenmore compatible with commercially available microscope equipment.

FIG. 9A schematically depicts an embodiment of the fabrication of ahorizontal MTF device using the protective film procedure in amulti-well dish with a zoomed in view of the film inside a well (not toscale).

FIG. 9B is a photo of a 24-well plate containing a checkered pattern ofbuffer with phenol red (grey) and without phenol red (white) used todetect leaks between wells.

FIG. 9C is a photo of a muscle thin film inside of a well of the 24-wellplate depicted in FIG. 9B.

FIG. 10 schematically depicts one embodiment of the fabrication of a twotissue type horizontal MTF device useful for a side-by-side assay.

FIG. 11 schematically depicts another embodiment of the fabrication of atwo tissue type horizontal MTF device useful for a side-by-side assay.

FIG. 12 contrasts the use of fluorescent microscopy and the use ofbrightfield stereo microscopy of vascular smooth muscle (VSM) horizontalMTFs during an assay of the invention as described in Example 5. The toprow depicts fluorescent microscopy photographs of VSM horizontal MTFscomprising fluorescent beads embedded in the PDMS film. The bottom rowshows brightfield stereo microscopy images of the same films. (A)Photographs taken 5 minutes after the start of the experiment, the filmsare slightly bent up from the glass due to the initial contraction ofVSM. (B) Photographs taken 20 minutes after addition of thevasoconstrictor (Endothilin-1), which causes the VSM to contract and thefilms to bend up from the glass. (C) Photographs taken 30 minutes afteraddition of a rho-kinase inhibitor, which causes the VSM to completelyrelax and the films to lie flat on the glass. The contrast of the use offluorescent microscopy shows that fluorescent images may be used toeliminate noise that may be present in regular brightfield images.

FIG. 13A is a top view schematic representation of one embodiment of thedevices of the invention depicting MTFs enclosed in individual chambersof a microfluidics device. Utilizing microfluidic principles of laminarflow and mixing, a small amount of nanoparticles or small molecules canbe diluted into a wide variety of concentrations in simultaneous assaysfor tissue function.

FIG. 13B is a side view schematic representation of one embodiment ofthe devices of the invention depicting MTFs enclosed in individualchambers of a microfluidics device. Utilizing microfluidic principles oflaminar flow and mixing, a small amount of nanoparticles or smallmolecules can be diluted into a wide variety of concentrations insimultaneous assays for tissue function.

FIG. 14 is a photograph of one embodiment of the devices of theinvention depicting a tri-laminate fluidic chamber comprising an MTF anduseful in the methods of the invention, such as, a contractility assay.The device is constructed from 1.5 mm PMMA. Number 1 glass cover slipscomprise the top and bottom layers. Muscular Thin Films (MTFs) were cutinto approximately 1 mm×3 mm cantilevers before assembling the device.

FIG. 15 depicts an anisotropic MTF in a microfluidic chamber exposed toa drug (circles) in diastole (top) and at peak systole (bottom).

FIG. 16 is a schematic representation of a gradient generationmicrofluidic device to assay drug-dose responses. The PDMS-based device(25 mm×75 mm), receives three fluids at left, the highest drug dose inthe top channel, a decreased drug concentration in the middle channel,and isotonic buffer in the bottom channel. The microfluidic devicesgenerate drug gradients which are then separated and transferred intowide (2.5 mm) channels to decrease fluid velocity in order to simplifyfluid dynamics calculations for horizontal MTF (hMTF) assays.

FIG. 17 is a schematic representation illustrating softlithography-based microfluidic fabrication of one embodiment of thedevices of the invention. A negative photoresist underneath a mask isexposed to UV light crosslinking the exposed photoresist. Theun-crosslinked photoresist is developed, leaving a negative mold. PDMSelastomer is poured into the photoresist/silicon mold and peeled awayafter curing. The patterned PDMS can be either bonded to another PDMSpattern or planar polymeric surface via plasma-treated surface covalentbonding.

FIG. 18 is a photograph of the top view of a microfluidic channel with ahMTF inside the channel. The inflow, outflow channel are outlined with adashed light gray line. The myocytes are paced inside the channel andthe diastolic and peak systolic positions of the hMTF are shown insidethe boxes, with the film edge tracked (light gray) and the film lengthoutlined (black). (B) Schematic showing the microfluidic channel with ahMTF inside, MTF film in medium gray, initial length outline in black,edge tracking in light gray, channel outline in light gray. (C) Filteredstress profile read-out from this film in kPa.

FIG. 19 schematically depicts an embodiment of the fabrication of a“shutter” horizontal MTF device in a multi-well dish. (A) A 96-wellplate with a photodiode array top. Each photodiode measures, as afunction of time, the amount of light emitted from each well, whichholds a single horizontal MTF. The readouts of the photodiodes aretransferred to a computer, where they are directly converted to ameasurement of the radius of curvature, and muscle layer stress. (B) Across-section of a single well with a “shutter design”. The bottom ofthe plate is made from glass that has been blacked out except for asmall area under the muscular thin film, which remains transparent tolight. The muscular thin films are made from non-transparentbio-compatible polymers, i.e. black PDMS. The walls of the plate aremade from black plastic, or other light absorbing material, to eliminatethe reflection of light. During contraction of the cells, the thin filmbends up from the glass uncovering a larger area of the transparentglass, allowing for a greater amount of light to pass through thechamber. The photodiode is located at the top of the well, where itcollets light throughout the contraction cycle. (C) The top view into asingle well of the “shutter system”. This illustrates that the island oftransparent glass has the same area as an unbent film (length a). As thefilm dissociates from the glass it bends up slightly during diastole,exposing some of the transparent glass. During the contraction the filmbends up more, exposing more of the transparent glass. The amount oflight reaching the photodiode will be proportional to the area of thewhite rectangle. As the width of the rectangle remains constant thesignal is proportional to w which is simply the length of the film minusthe x-projection. Therefore the signal transmitted from the photodiodesis proportional to the x-projection. Example 2 and FIG. 7C describe howto calculate the radius of curvature from measurements of thex-projection. However, the use of a photodiode array dispenses with theneed for image analysis as the x-projection is a direct readout. Thisallows for this device to be an automated, high-throughput, simultaneouscontraction measuring device.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods and devices for multiplex and highthroughput measurements of various properties of in vitro tissueconstructs, e.g., for simultaneous or high-speed serial analysis ofnumerous samples. The devices and methods of the present invention canbe used to measure muscle activities or functions, e.g., biomechanicalforces that result from stimuli that include, but are not limited to,muscle cell contraction, osmotic swelling, structural remodeling andtissue level pre-stress. The devices and methods of the presentinvention may also be used for the evaluation of muscle activities orfunctions, e.g., electrophysiological responses, in a non-invasivemanner, for example, in a manner that avoids cell damage. The devicesand methods of the present invention are also useful for investigatingmuscle cell developmental biology and disease pathology, as well as indrug discovery and toxicity testing.

I. Devices of the Invention and Methods of Production of the Same

In one aspect, the present invention provides devices, e.g., devices formeasuring a contractile function, which comprise a solid supportstructure, a plurality of muscle thin films (MTFs) adhered to the solidsupport structure, wherein the plurality of muscle thin films eachcomprise a flexible polymer layer and a population of isolated cellsseeded on the flexible polymer layer in a predetermined pattern, andwherein the cells form a tissue structure which can perform acontractile function. The MTFs may be adhered to the solid supportstructure directly or indirectly, e.g., via the use of a post (e.g., themiddle of an MTF is adhered to the post, as described in further detailbelow). One end (as in FIG. 6A) or both ends of the MTFs may be adheredto the solid support structure.

In one embodiment, the MTFs that are used in the devices and methods ofthe present invention may be prepared as described in PCT PublicationNo. WO 2008/051265, the entire contents of which are incorporated hereinby reference. Briefly, substrates or devices for use in the methods ofthe invention are fabricated as a rigid base material coated with asacrificial polymer layer; a flexible polymer layer is temporarilybonded to the rigid base material via the sacrificial polymer layer, andan engineered surface chemistry is provided on the flexible polymerlayer to enhance or inhibit cell and/or protein adhesion. Cells areseeded onto the flexible polymer layer, and cultured to form a tissuecomprising, for example, patterned anisotropic myocardium. A desiredshape of the flexible polymer layer can then be cut; and the flexiblefilm, including the polymer layer and tissue, can be peeled off with apair of tweezers as the sacrificial polymer layer dissolves to releasethe flexible polymer layer, to produce a free-standing film.

The base layer may be formed of a rigid or semi-rigid material, such asa plastic, metal, ceramic, or a combination thereof. In one embodiment,the base layer is transparent so as to facilitate observation. Inanother embodiment, the base layer is opaque (e.g., light-absorbing). Inone embodiment, a portion of the base layer is transparent (i.e., aportion underneath a portion of the MTF) and the remaining portion isopaque. In yet another embodiment, the base layer is translucent. Thebase layer is ideally biologically inert, has low friction with thetissues and does not interact (e.g., chemically) with the tissues.Examples of materials that can be used to form the base layer includepolystyrene, polycarbonate, polytetrafluoroethylene (PTFE), polyethyleneterephthalate, quartz, silicon, and glass. In one embodiment, the baselayer is a silicon wafer, a glass cover slip, a multi-well plate, atissue culture plate, a Petri dish, or a microfluidic chamber.

In one embodiment, the base layer and the solid support structure arethe same. For example, as described below, a MTF may be fabricated on,for example, a glass cover-slip (the base layer) and subsequent tocoating a flexible polymer layer ont the sacrificial polymer layer, therigid base material is cut into sections. Such sections may be placedin, for example, a multi-well plate or a microfluidic chamber.

In another embodiment, the solid support structure and the base layerare different. For example, as described below, a MTF may be fabricatedon, for example, a glass cover-slip (the base layer) which issubsequently cut into strips and applied to a solid support structure,such as a post adhered to a Petri-dish or a microfluidic chamber.

The sacrificial polymer layer is deposited on the base layer, i.e., isplaced or applied onto the base layer. Depositing may include, but isnot limited to, spraying, dip casting, and spin-coating. The sacrificialpolymer layer may be deposited on substantially the entire surface oronly a portion of the surface of the base layer.

In one embodiment, spin-coating is used to deposit the sacrificialpolymer layer on the base material. “Spin-coating” is a process whereinthe base layer is mounted to a chuck under vacuum and is rotated to spinthe base layer about its axis of symmetry while a liquid or semi-liquidsubstance, e.g. a polymer, is dripped onto the base layer. Centrifugalforce generated by the spin causes the liquid or semi-liquid substanceto spread substantially evenly across the surface of the base layer.

In one embodiment, the sacrificial polymer is a thermally sensitivepolymer that can be melted or dissolved to release the flexible polymerlayer. For example, linear non-cross-linked poly(N-Isopropylacrylamide)(PIPAAM), which is a solid when dehydrated or at about 37° C., whereinthe polymer is hydrated, but relatively hydrophobic. When thetemperature of the polymer is dropped to about 35° C. or less, whereinthe polymer is hydrated, but relatively hydrophilic, the polymerliquefies, thereby releasing the patterned flexible polymer layer(Feinberg et al., Science 317:1366-1370, 2007).

In another embodiment, the sacrificial polymer becomes hydrophilic whenthe temperature is lowered, thereby releasing hydrophobic coatings. Forexample, the sacrificial polymer can be hydrated, cross-linked PIPAAM,which is hydrophobic at about 37° C. and hydrophilic at about 35° C. orless (e.g., about 35° C. to about 32° C.). In yet another embodiment,the sacrificial polymer is an electrically actuated polymer that becomeshydrophilic upon application of an electric potential and releases ahydrophobic structure coated thereon. Examples of such a polymer includepoly(pyrrole)s, which are relatively hydrophobic when oxidized andhydrophilic when reduced. Other examples of polymers that can beelectrically actuated include poly(acetylene)s, poly(thiophene)s,poly(aniline)s, poly(fluorene)s, poly(3-hexylthiophene),polynaphthalenes, poly(p-phenylene sulfide), and poly(para-phenylenevinylene)s. In another embodiment, the sacrificial polymer is adegradable biopolymer that can be dissolved to release a structurecoated thereon. For example, the polymer (e.g., polylactic acid,polyglycolic acid, poly(lactic-glycolic) acid copolymers, or nylons)undergoes time-dependent degradation by hydrolysis or by enzymaticaction (e.g., fibrin degradation by plasmin, collagen degradation bycollagenase, fibronectin degradation by matrix metalloproteinase).

In one embodiment, the sacrificial polymer is an ultra-hydrophobicpolymer with a surface energy lower than the flexible polymer layeradhered to it. In this case, mild mechanical agitation will “pop” thepatterned flexible polymer layer off of sacrificial polymer layer.Examples of such a polymer include but are not limited to alkylsilanes(octadecyltrichiorosilane and isobutyltrimethoxysilane),fluoroalkylsilanes (tridecafluorotetrahydrooctyltrichiorosilane,trifluoropropyltrichiorosilane andheptadecafluorotetrahydrodecyltrichlorosilane), silicones(methyihydrosiloxane-dimethylsiloxane copolymer, hydride terminatedpolydimethylsiloxane, trimethylsiloxy terminated polydimethylsiloxaneand diacetoxymethyl terminated polydimethylsiloxane), fluorinatedpolymers (polytetrafluoroethylene, perfluoroalkoxy and fluorinatedethylene propylene). For example, the base material can be a glass coverslip coated with a sacrificial polymer layer of PIPAAM.

Examples of the elastomers that can be used to form the flexible polymerlayer include polydimethylsiloxane (PDMS) and polyurethane. In oneembodiment, the PDMS, once cured is opaque (e.g., light-absorbing). Inother embodiments, thermoplastic or thermosetting polymers are used toform the flexible polymer layer. Alternative non-degradable polymersinclude polyurethanes, silicone-urethane copolymers, carbonate-urethanecopolymers, polyisoprene, polybutadiene, copolymer of polystyrene andpolybutadiene, chloroprene rubber, Polyacrylic rubber (ACM, ABR),Fluorosilicone Rubber (FVMQ), Fluoroelastomers, Perfluoroelastomers,Tetrafluoro ethylene/propylene rubbers (FEPM) and Ethylene vinyl acetate(EVA). In still other embodiments, biopolymers, such as collagens,elastins, polysaccharides, and other extracellular matrix proteins, areused to form the flexible polymer layer. Suitable biodegradableelastomers include hydrogels, elastin-like peptides,polyhydroxyalkanoates and poly(glycerol-sebecate). Suitablenon-elastomer, biodegrable polymers include polylactic acid,polyglycolic acid, poly lactic glycolic acid copolymers.

In one embodiment, the flexible polymer layer comprisespolydimethylsiloxane (PDMS). Thickness of the PDMS layer can becontrolled by the viscosity of the prepolymer and by the spin-coatingspeed, ranging from 14 to 60 nm thick after cure. The viscosity of theprepolymer increases as the cross-link density increases. This change inviscosity between mixing and gelation can be utilized to spin-coatdifferent thicknesses of flexible polymer layers. Alternatively thespin-coating speed can be increased to create thinner polymer layers.After spin-coating, the resulting polymer scaffolds are either fullycured at room temperature (generally, about 22° C.) or at 65° C.

In one embodiment, polymeric fibers prepared as described in U.S.Provisional Application No. 61/177,894, entitled “Methods and Devicesfor the fabrication of 3D Polymeric Fibers”, filed, May 12, 2009, (theentire contents of which are incorporated herein by reference) may beused as for the sacrificial polymer layer and/or the flexible polymerlayer.

In one embodiment, fluorescent beads, e.g., fluorospheres, are mixedwith the flexible polymer layer prior to spin-coating the flexiblepolymer layer onto the sacrificial polymer layer.

The flexible polymer layer is then uniformly or selectively patternedwith engineered surface chemistry to elicit (or inhibit) specific cellgrowth and function. The engineered surface chemistry can be providedvia exposure to ultraviolet radiation or ozone or via acid or base washor plasma treatment to increase the hydrophilicity of the surface.

A specific biopolymer (or combination of biopolymers) may be selected torecruit different integrins, or an engineered surface chemistry may befabricated on the flexible polymer layer to enhance or inhibit celland/or protein adhesion. The specific type of biopolymer used andgeometric spacing of the patterning will vary with the application. Inone embodiment, the engineered surface chemistry comprises a biopolymer,such as an ECM protein, to pattern specific cell types. The ECM maycomprise fibronectin, laminin, one or more collagens, fibrin,fibrinogen, or combinations thereof. In one embodiment, the ECM is notuniformly distributed on the surface of the flexible polymer, but ratheris patterned spatially using techniques including, but not limited to,soft lithography, self assembly, printed on the solid support structurewith a polydimethylsiloxane stamp, vapor deposition, andphotolithography. In one embodiment, the methods of the inventionfurther comprise printing multiple biopolymer structures, e.g., the sameor different, with successive, stacked printings. Additional suitablesurface chemistries are provided in PCT Publication No. WO 2008/051265.

In one embodiment of the invention, a MTF is engineered usingalternating high density lines of ECM protein with either low densityECM protein or a chemical that prevents protein adhesion (e.g.,Pluronics F127). The spacing of these lines is typically 20 μm width at20 μm spacing, (Feinberg, Science 317:1366-1370, 2007), however, thewidth and spacing may be altered to change the alignment. Changes inalignment in turn affect anisotropy and anisotropy ratio of the actionpotential propagation. The width and spacing of the ECM lines may bevaried over the range from about 0.1 μm to about 1000 μm, from about 1μm to about 500 μm, from about 1 μm to 250 μm, from about 1 μm to 100μm, from about 1 μm to 90 μm, from about 1 μm to 80 μm, from about 1 μmto 70 μm, from about 1 μm to 60 μm, from about 1 μm to 50 μm, from about1 μm to 40 μm, from about 1 μm to 30 μm, from about 1 μm to 20 μm, fromabout 1 μm to 10 μm, from about 2 μm to 100 μm, from about 2 μm to 90μm, from about 2 μm to 80 μm, from about 2 μm to 70 μm, from about 2 μmto 60 μm, from about 2 μm to 50 μm, from about 2 μm to 40 μm, from about2 μm to 30 μm, from about 2 μm to 20 μm, from about 2 μm to 10 μm, fromabout 1 μm to 100 μm, from about 5 μm to about 100 μm, from about 5 μmto about 90 μm, from about 5 μm to about 80 μm, from about 5 μm to about70 μm, from about 5 μm to about 60 μm, from about 5 μm to about 50 μm,from about 5 μm to about 40 μm, from about 5 μm to about 30 μm, fromabout 5 μm to about 20 μm, and from about 5 μm to about 20 μm. The widthand spacing of the ECM lines can be equivalent or disparate. Forexample, both the width and spacing can be about 0.1, about 0.2, about0.25, about 5, about 0.75, about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 5, about 16, about 17, about 18, about 19, or about20 μm. In other embodiments, the width can be about 0.1, about 0.2,about 0.25, about 5, about 0.75, about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, or about 10 μm and thespacing can be about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 5, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, about 31, about 32, about 33, about 34, about 35,about 36, about 37, about 38, about 39, or about 40 μm. Conversely, thewidth can be about 1, about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 5, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, about 31, about 32, about 33, about 34, about 35,about 36, about 37, about 38, about 39, or about 40 μm and the spacingcan be about 0.1, about 0.2, about 0.25, about 5, about 0.75, about 1,about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,or about 10 μm. Typically the patterned ECM lines are parallel to oneanother, but they may also be at angles to one another at about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°. In oneembodiment, the patterned ECM lines are parallel to one another atangles ranging from about 1° to about 90°. In another embodiment, thepatterned ECM lines are parallel to one another at angles ranging fromabout 5° to about 45°. The angle between the patterned lines of ECMprotein controls the directionality of action potential propagation. Thewidth of the MTF itself can be tapered to control the direction ofaction potential propagation. For example, a wide MTF strip that tapersto a narrow strip will propagate an action potential from the wide tothe narrow direction, but not in the opposite direction. Values andranges intermediate to the above-recited values and ranges are alsocontemplated by the present invention.

In another embodiment, a MTF is engineered using stretching of, e.g.,the flexible polymer layer during tissue formation. In one embodiment,the stretching is cyclic stretching. In another embodiment, thestretching is sustained stretching. In one embodiment, the flexiblepolymer layer is stretched at an appropriate time after cell seedingthat is based on the type(s) of cells seeded. In one embodiment, theflexible polymer layer is stretched at about minutes, hours, or daysafter cell seeding onto a patterned flexible polymer layer. In oneembodiment, the flexible polymer layer is stretched at about 0, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, orabout 3.0 hours after cell seeding onto a patterned flexible polymerlayer. In one embodiment, the flexible polymer layer is patternedisotropically. Stretching, therefore, results in the formation ofanisotropic tissue, the anisotropy of which is in the direction of thestretch. In another embodiment, the flexible polymer layer is patternedanistropically and stretching enhances the anisotropy of the tissueformed.

In one embodiment, the flexible polymer layer is stretched using about a0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5.0, 5.5,or about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10.0 Hertz(Hz) cyclic stretch. In one embodiment, the flexible polymer layer isstretched using about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5,14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5,or about 20.0% strength sustained stretch.

To attach cells, substrates are placed in culture with a cell suspensionallowing the cells to settle and adhere to the surface. In the case ofan adhesive surface treatment, cells bind to the material in a mannerdictated by the surface chemistry. For patterned chemistry, cellsrespond to patterning in terms of maturation, growth and function.Examples of cell types that may be used include contractile cells, suchas, but not limited to, vascular smooth muscle cells, vascularendothelial cells, myocytes (e.g., cardiac myocytes), skeletal muscle,myofibroblasts, airway smooth muscle cells and cells that willdifferentiate into contractile cells (e.g., stem cells, e.g., embryonicstem cells or adult stem cells, progenitor cells or satellite cells).

The term “progenitor cell” is used herein to refer to cells that have acellular phenotype that is more primitive (e.g., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells also have significant or veryhigh proliferative potential. Progenitor cells can give rise to multipledistinct differentiated cell types or to a single differentiated celltype, depending on the developmental pathway and on the environment inwhich the cells develop and differentiate.

The term “progenitor cell” is used herein synonymously with “stem cell.”

The term “stem cell” as used herein, refers to an undifferentiated cellwhich is capable of proliferation and giving rise to more progenitorcells having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated, or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers to a subset ofprogenitors that have the capacity or potential, under particularcircumstances, to differentiate to a more specialized or differentiatedphenotype, and which retains the capacity, under certain circumstances,to proliferate without substantially differentiating. In one embodiment,the term stem cell refers generally to a naturally occurring mother cellwhose descendants (progeny) specialize, often in different directions,by differentiation, e.g., by acquiring completely individual characters,as occurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell which itself is derived from a multipotent cell, and soon. While each of these multipotent cells may be considered stem cells,the range of cell types each can give rise to may vary considerably.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. In manybiological instances, stem cells are also “multipotent” because they canproduce progeny of more than one distinct cell type, but this is notrequired for “stem-ness.” Self-renewal is the other classical part ofthe stem cell definition. In theory, self-renewal can occur by either oftwo major mechanisms. Stem cells may divide asymmetrically, with onedaughter retaining the stem state and the other daughter expressing somedistinct other specific function and phenotype. Alternatively, some ofthe stem cells in a population can divide symmetrically into two stems,thus maintaining some stem cells in the population as a whole, whileother cells in the population give rise to differentiated progeny only.Formally, it is possible that cells that begin as stem cells mightproceed toward a differentiated phenotype, but then “reverse” andre-express the stem cell phenotype, a term often referred to as“dedifferentiation” or “reprogramming” or “retrodifferentiation”.

The term “embryonic stem cell” is used to refer to the pluripotent stemcells of the inner cell mass of the embryonic blastocyst (see U.S. Pat.Nos. 5,843,780, 6,200,806, the contents of which are incorporated hereinby reference). Such cells can similarly be obtained from the inner cellmass of blastocysts derived from somatic cell nuclear transfer (see, forexample, U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970, which areincorporated herein by reference). The distinguishing characteristics ofan embryonic stem cell define an embryonic stem cell phenotype.Accordingly, a cell has the phenotype of an embryonic stem cell if itpossesses one or more of the unique characteristics of an embryonic stemcell such that that cell can be distinguished from other cells.Exemplary distinguishing embryonic stem cell characteristics include,without limitation, gene expression profile, proliferative capacity,differentiation capacity, karyotype, responsiveness to particularculture conditions, and the like.

The term “adult stem cell” or “ASC” is used to refer to any multipotentstem cell derived from non-embryonic tissue, including fetal, juvenile,and adult tissue. Stem cells have been isolated from a wide variety ofadult tissues including blood, bone marrow, brain, olfactory epithelium,skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stemcells can be characterized based on gene expression, factorresponsiveness, and morphology in culture. Exemplary adult stem cellsinclude neural stem cells, neural crest stem cells, mesenchymal stemcells, hematopoietic stem cells, and pancreatic stem cells.

In one embodiment, progenitor cells suitable for use in the claimeddevices and methods are Committed Ventricular Progenitor (CVP) cells asdescribed in PCT Application No. PCT/US09/XXXXX, entitled “TissueEngineered Mycocardium and Methods of Productions and Uses Thereof”,filed Sep. 28, 2009, the entire contents of which are incorporatedherein by reference.

The cells on the substrates are cultured in an incubator underphysiologic conditions (e.g., at 37° C.) until the cells form atwo-dimensional (2D) tissue (i.e., a layer of cells that is less thanabout 200 microns thick, or, in particular embodiments, less than about100 microns thick, less than about 50 microns thick, or even just amonolayer of cells), the anisotropy or isotropy of which is determinedby the engineered surface chemistry.

Any appropriate cell culture method may be used to establish the tissueon the polymer surface. The seeding density of the cells will varydepending on the cell size and cell type, but can easily be determinedby methods known in the art. In one embodiment, cardiac myocytes areseeded at a density of between about 1×10⁵ to about 6×10⁵ cells/cm², orat a density of about 1×10⁴, about 2×10⁴, about 3×10⁴, about 4×10⁴,about 5×10⁴, about 6×10⁴, about 7×10⁴, about 8×10⁴, about 9×10⁴, about1×10⁵, about 1.5×10⁵, about 2×10⁵, about 2.5×10⁵, about 3×10⁵, about3.5×10⁵, about 4×10⁵, about 4.5×10⁵, about 5×10⁵, about 5.5×10⁵, about6×10⁵, about 6.5×10⁵, about 7×10⁵, about 7.5×10⁵, about 8×10⁵, about8.5×10⁵, about 9×10⁵, about 9.5×10⁵, about 1×10⁶, about 1.5×10⁶, about2×10⁶, about 2.5×10⁶, about 3×10⁶, about 3.5×10⁶, about 4×10⁶, about4.5×10⁶, about 5×10⁶, about 5.5×10⁶, about 6×10⁶, about 6.5×10⁶, about7×10⁶, about 7.5×10⁶, about 8×10⁶, about 8.5×10⁶, about 9×10⁶, or about9.5×10⁶. Values and ranges intermediate to the above-recited values andranges are also contemplated by the present invention.

In one embodiment, cardiac myocytes are co-cultured with neurons toprepare innervated engineered tissue comprising pacemaking cells, and/orto accelerate the maturation of the cultured cells as described in U.S.Provisional Application No. 61/306,736, the entire contents of which areincorporated herein by reference.

In one embodiment, a specific shape (e.g., a rectangular strip) is cutin the flexible polymer film using a scalpel, razor blade, punch, die orlaser. The sacrificial layer is then dissolved or actuated to releasethe flexible polymer from the rigid base (e.g., by dropping thetemperature below 35° C.); and the cut-out shape then floats free or isgently peeled off. The bending stiffness of the thin films along anygiven axis, much like a cantilever, increases with the elastic modulus,thickness and width while decreasing with length.

Suitable support structures for embodiments of the present inventioninclude, for example, Petri dishes, cover-slips (circular orrectangular), strips of glass, glass slides, multi-well plates,microfluidic chambers, and microfluidic devices. The support structureis ideally biologically inert, it has low friction with the films and itdoes not interact (e.g., chemically) with the films. Examples ofmaterials that can be used to form the support structure includepolystyrene, polycarbonate, polytetrafluoroethylene (PTFE), polyethyleneterephthalate, quartz, silicon (e.g., silicon wafers) and glass. In oneembodiment, the support structure is transparent. In another embodiment,the support structure is opaque (e.g., light-absorbing). In oneembodiment, a portion of the base layer is transparent (i.e., a portionunderneath a portion of the MTF) and the remaining portion is opaque. Inyet another embodiment, the base layer is translucent.

In one embodiment the base layer and the solid substrate are fabricatedfrom the same material. In another embodiment, the base layer and thesolid support are fabricated from different materials.

Examples of suitable materials for a co-culture device include, but arenot limited to, tissue culture polystyrene, acid washed glass,extracellular matrix (e.g., collagen, fibronectin, fibrin, laminin)coated glass or polymer (e.g., PET, nylon), poly L-lysine coated glassor polymer.

In the embodiments of the invention in which the MTFs are attached tothe solid support structure indirectly, via a post (as in FIGS. 1-3),the posts may be formed from a rigid material, such as polystyrene,stainless steel, polytetrafluoroethylene (PTFE) or a cactus needle. Thepost may also be adhesive to the thin film. In some embodiments, thepost and film are mechanically adhesive (e.g., the film and post areclipped to each other).

In other embodiments, the post and film are chemically adhesive. Forexample, the post can be coated with adhesive glue. Alternatively, thepost is coated or formed from a material that interacts with theflexible polymer layer of the film. For example, as shown in FIG. 3, thePDMS of the flexible polymer layer of the thin film adheres to a PTFEpost through a hydrophobic-hydrophobic interaction. In otherembodiments, the post may be coated with a material (e.g., polyL-lysine, collagen, fibronectin, fibrin, laminin) that binds to the celllayer of the thin film.

The appended Figures depict various embodiments of the devices of thepresent invention. For example, FIG. 1 depicts a device of the inventionin which a homogeneous cell population is used. As shown in FIGS. 1A and1B, schematic perspective and top views respectively, the systemincludes a solid support structure, such as a Petri dish, with postsaffixed to it and substantially perpendicular to the base of the solidsupport structure. Thin films are adhered to the posts. A photograph ofa device according to this embodiment is shown in FIG. 1C.

FIG. 2 depicts a device of FIG. 1 that further includes a co-culturedevice, e.g., slide, that can be inserted into the assay dish. As shownin FIG. 2, the system includes a ring on which additional cells can beseeded, for the study of cell-cell interactions such as, for example,paracrine signaling. Cell-cell interactions, such as paracrine signalingcan be studied by using films cultured with different cell types (e.g.,vascular smooth muscle and cardiac myocytes) together. The use of thinfilms with different cell types together with a co-culture device allowsfor studies of three or more cell types.

The devices of FIGS. 1 and 2 can be used as follows: When a co-culturedevice with secondary cells (e.g., aortic endothelial cells) is used, itis placed in the device, e.g., dish, in a buffered medium. Thin filmsare prepared and cut into strips, as discussed below or as described inPCT Publication No. WO 2008/051265. The strips are transferred to thedevice and are adhered to the posts such that the plane of primarycurvature is parallel with the plane of the device, e.g., Petri dishbase. A stimulus is applied to the films to cause stress in the celllayer. The curvature of the films is recorded and cell stress iscalculated. A fluid perfusion system can be used to wash out testcompounds that are being screened in a high throughput assay or torefresh the culture medium. A typical experiment using the device ofFIG. 1 is shown in FIG. 4, where rectangular-shaped muscular thin filmswith vascular smooth muscle anisotropically aligned along their lengthswere used, and the deformation of the muscular thin films with timeafter treatment with endothelin-1 was measured.

FIG. 5 schematically depicts one embodiment for the fabrication and useof a multi-well based device of the invention. According to thisembodiment, an array of thin strips is created on a solid supportstructure. Rings are placed on the solid support structure creating amulti-well plate (e.g., about 8-, about 12-, about 16-, about 20-, about24-, about 28-, about 32-about 36-, about 40, about 44, about 48-, about96-, about 192-, about 384-wells) and isolating the strips. Cells arethen seeded and cultured onto the strips to form tissue structures asdescribed below or as described in PCT Publication No. WO 2008/051265.In one embodiment, cells are cultured in the presence of a fluorophoreor fluorescent beads. One end of the strips is optionally detached fromthe solid support structure and released to form structures, e.g.,cantilevers, which are free to deform when the tissue structurescontract. The deformation (i.e., contractility) of the MTFs may berecorded, e.g., as depicted in FIG. 6. In the embodiment depicted inFIG. 6, contractility may be observed (and optionally recorded) using amicroscope, which looks at one strip at a time while it scans acrossmultiple samples (see FIG. 6B). In one embodiment of the invention,multiple strips are observed simultaneously (see FIG. 6A). Optionally, alens is integrated into the platform. Changes in the curvature of thefilms are observed and the optical image is converted to a numericalvalue that corresponds to the curvature of the film. In one embodiment,a movie of MTF contractions in a multi-well dish is acquired (e.g.,images are obtained in series). Images are processed and a mechanicalanalysis is optionally carried out to evaluate contractility. The outputmay be traction as a function of standard metrics such as peak systolicstress, peak upstroke power, upstroke time, and relaxation time.

Alternative ways of measuring bending of MTFs include, e.g., (i) using alaser bounced off of the thin film to record movement, (ii) using anintegrated piezoelectric film in the MTF and recording a change involtage during bending, (iii) integrating magnetic particles in the MTFand measuring the change in magnetic field during bending, (iv) placinga lens in the bottom of each well and simultaneously projecting multiplewells onto a single detector (e.g., camera, CCD or CMOS) at one time,(v) using a single capture device to sequentially record each well (see,e.g., FIG. 6(2)), e.g., the capture device is placed on an automatedmotorized stage. Finally, the measured bending information (e.g.,digital image or voltage) is converted into force, frequency and othercontractility metrics.

In another embodiment, as depicted in FIGS. 7A, 7B, and 7C, an MTFdevice is constructed horizontally rather than vertically such thathandling of the MTF is not necessary allowing for increased throughputof MTF production. More specifically and similar to the MTF fabricationprocess described in WO 2008/051265, a substrate or device is fabricatedas a rigid base material which is coated partially, i.e., all of theedges of the base material are not covered with a sacrificial polymerlayer; a flexible polymer layer is temporarily bonded to the rigid basematerial via the sacrificial polymer layer, and an engineered surfacechemistry is provided on the flexible polymer layer to enhance orinhibit cell and/or protein adhesion. Cells are seeded onto the flexiblepolymer layer and cultured to form a tissue. The formed tissue is thenplaced at a lower temperature (e.g., 35° C.)

In order to create the horizontal MTFs, sections of the flexible polymerlayer can be cut and removed such that strips of the flexible film,including the polymer layer and tissue remain secured at their base tothe base material and act as a hinge. This method allows the MTFs tocurve upward off the base layer, i.e., to curve upward from the viewing(horizontal plane), as compared to the MTFs described above in which theMTF bends in the viewing plane) when stimulated to contract (see, e.g.,FIGS. 1-3). In this embodiment, individual MTFs (e.g., 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or moreMTFs) can be prepared on a single solid support structure, e.g., a glasscover slip (round or rectangular), a Petri dish, a glass slide, stripsof glass, or a multi-well plate. The functional properties of theseMTFs, e.g., the contractility of these MTFs, may be determined asdescribed above for a vertical MTF.

In another embodiment, horizontal MTFs are fabricated as depicted inFIGS. 8A and 8B. More specifically, a protective film, e.g., staticvinyl sheet or tape, e.g., adhesive tape, is applied to one or moreportions of a rigid base material in order to prevent adherence of asacrificial polymer to the rigid base material. The protective film maybe applied to the rigid base material by, e.g., contacting the rigidbase material with a liquid prior to applying the protective film togenerate a liquid interface (e.g., any solvent that does not leavebehind a residue on the rigid base material, e.g., ethanol) between therigid base material and the protective film, and removing the excessliquid. In one embodiment, one or more portions of the top of the rigidbase material (i.e., where the MTF will be formed) is coated with aprotective film. In another embodiment, one or more portions, or all ofthe bottom (i.e., where the MTF will not be formed) of the rigid basematerial is coated with a protective film. In another embodiment, one ormore portions of the top of the rigid base material and one or moreportions or all of the bottom of the rigid base material are coated witha protective film.

In one embodiment, one or more sections of the protective film on thetop surface of the rigid base material are cut and removed, therebycreating islands of rigid base material. The rigid base materialpartially coated with the protective film is then coated with asacrificial polymer layer and the remaining protective film on the topof the rigid base material is removed. Subsequently, a flexible polymerlayer is temporarily bonded to the rigid base material via thesacrificial polymer layer. If used, the bottom protective layer is thenremoved. Next, an engineered surface chemistry is provided on theflexible polymer layer to enhance or inhibit cell and/or proteinadhesion, cells are seeded onto the flexible polymer layer and culturedto form a tissue, as described above. The formed tissue is then placedat a lower temperature (e.g., 35° C.) to dissolve the sacrificialpolymer layer and sections of the flexible polymer layer (correspondingo the islands) can be cut to create the horizontal MTFs.

In one embodiment, the methods for fabricating a horizontal MTF (using aprotective film described above), further comprise attaching amulti-well plate skeleton to the rigid base material subsequent topatterning the flexible polymer layer with an engineered surfacechemistry and prior to cell seeding (see, e.g., FIG. 9A).

In one embodiment, a device comprising a horizontal MTF and a multi-wellplate further comprises a photodiode array (see, e.g., FIG. 19).

In one embodiment, as described above for a vertical MTF, the solidsupport structure may further comprise an optical signal capture deviceand an image processing software to calculate change in an opticalsignal. The optical signal capture device may further include fiberoptic cables in contact with the device and/or a computer processor incontact with the device.

In one embodiment, an electrode is in contact with the device.

In certain embodiments of the invention, prior to patterning theflexible polymer layer with an engineered surface chemistry, the rigidbase material coated with a sacrificial polymer layer and a flexiblepolymer layer, are divided, e.g., are cut, into portions, i.e.,separate, individual devices (see, FIGS. 10 and 11). Such devices areuseful for “side-by-side” assays that can be used, e.g., toqualitatively compare contractions of two tissue types, to compare theeffect of one tissue response in proximity to another tissue, to comparebiomechanical measurements of myocyte contraction properties, e.g., inresponse to various mechanical or chemical stimuli, to compare theeffect of various patterning on tissue contractility, to comparedifferent types of cells, or as an appropriate control. In otherembodiments, the device is divided into individual devices prior to cellseeding and subsequent to patterning the flexible polymer layer with anengineered surface chemistry.

In one embodiment, devices that are divided subsequent to patterning theflexible polymer layer with an engineered surface chemistry may befurther contacted with the same or different engineered surfacechemistry to elicit or inhibit specific cell growth and/or function. Inone embodiment, an individual device is combined with (e.g., placed inphysical proximity to) one or more other individual devices subsequentto cell seeding such that the two devices share the same media and/ortest compound and/or can send a paracrine signal. In one embodiment, twoor more devices placed in physical proximity to each other are separatedby a membrane which allows molecules of a certain size to pass through.In another embodiment, two or more devices are combined by a channelsuch that they share the same media and/or test compound but cannot senda paracrine signal.

In another embodiment, the invention provides a microfluidics devicecomprising a solid support structure which comprises a plurality MTF(i.e., the device comprises a plurality of microfluidic chambers eachcomprising a MTF), such as depicted in FIGS. 13, 14, 16, and 18B anddescribed in, for example, PCT Publication Nos. WO 2010/042284, WO2007/044888, and WO 2010/041230, the entire contents of each of whichare incorporated herein by reference. In one embodiment, the pluralityof microfluidic chambers comprising a MTF is operably connected to twoor more inlet microchannels each comprising a valve, such as describedin, for example, WO 2007/044888, to regulate flow, and two or moreoutlet microchannels.

In one embodiment, the two or more inlet microchannels comprise one ormore mixing chambers (a section of the inlet microchannel that generatesturbidity). Such devices may have 2-1002 microchambers comprising a MTF,and 2, 3, 4, 5, 6, 7, 8, 9, or 10 inlet microchannels, each with avalve. Such devices may have from 1-1000 mixing chambers. Such devicesare useful for generating concentration gradients of a test compound toperform a dose response assay with the test compound. The number ofconcentrations of the test compound that may be produced in such adevice is dependent on the number of mixing chambers.

In another embodiment, the plurality of microfluidic chambers comprisinga MTF is operably connected to one or more inlet ports and does notcomprise a mixing chamber. Such devices may comprise 1-1000 inlet portsand 1-1000 microchambers comprising a MTF. Such devices are also usefulfor performing a dose response assay with a test compound, however thevarious drug concentrations must be pre-mixed and introduced intoaninlet port separately.

In one embodiment, the microfluidics devices of the invention furtheroptionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)collection ports.

Fluid may be moved through the microfluidics devices by any suitablemeans, such as electrochemical or pressure-driven means.

A microfluidic chamber and a microfluidic channel may be fabricated intoone or more materials including but not limited to, Polydimethylsiloxane(PDMS), polyurethanes, other elastomers, thermoplastics (e.g. polymethylmethacrylate (PMMA), polyethylene, polyethylene terephthalate,polystyrene), epoxies and other thermosets, silicon, silicon dioxide,and indium tin oxide (ITO).

Any suitable method may be used to fabricate a microfluidic channeland/or chamber, such as, for example, micromachining, injection molding,laser etching, laser cutting, and soft lithography. In one embodiment,an electrode is fabricated into a chamber using a non-reactive metal,such as, platinum, gold, and indium tin oxide.

A MTF suitable for use in a microfluidics device may be fabricated asdescribed herein and in, for example PCT Publication No. WO 2008/051265,and cut into suitably sized strips. Alternatively, horizontal polymericthin films may be fabricated as described herein. Cells may be seededprior to assembly of the thin film into a microfluidics device and/ormay be seeded subsequent to full assembly of the device. In addition, apolymeric thin film may be pre-fabricated into a chamber of themicrofluidics device, or may be added to a chamber of the device afterthe device is fabricated.

The benefits of such a device for use in the methods of the inventioninclude, for example, creation of a microenvironment that more closelyresembles an in vivo fluidic microenvironment, increasing the number ofassays that may be performed simultaneously while decreasing the amountof test compound required, ability to create a wide range of testcompound concentrations for simultaneous assaying, and the ability tomaintain MTFs in culture for up to one month in culture (depending onthe tissue type).

In yet other embodiments of the invention, MTFs may be free floating. Inone embodiment, MTFs are separated from the well edges by 1-2 lengths ofthe MTF.

In the embodiments of the invention where the solid support structure isa multi-well plate, each well may contain one MTF, two MTFs, or multipleMTFs.

In one embodiment, fluorescent beads, e.g., fluorospheres, are mixedwith the flexible polymer layer prior to spin-coating the flexiblepolymer layer onto the sacrificial polymer layer. The addition of suchbeads may enhance data capture.

In certain embodiments of the invention, e.g., for evaluation ofelectrophysiological activities, cells are cultured in the presence of afluorophor such as a voltage-sensitive dye or an ion-sensitive dye. Forexample, the voltage-sensitive dye is an electrochromic dye such as astyryl dye or a merocyanine dye. Exemplary electrochromic dyes includeRH-421 or di-4-ANEPPS. Ion-sensitive, e.g., calcium sensitive dyes,include aequorin, Fluo3, and Rhod2. For simultaneous measurements ofaction potentials and intracellular calcium, the following exemplary dyepairs are used: di-2-ANEPEQ and calcium green; di-4-ANEPPS and Indo-1;di-4-ANEPPS and Fluo-4; RH237 and Rhod2; and, RH-237 and Fluo-3/4.

In such embodiments, the device includes MTFs grown in multi-well, e.g.,2-8-, 12-, 16-, 20-, 24-, 28-, 32-36-, 40, 44, 48-, 96-, 192-, 384-well,plates prepared as described herein. An inverted microscope orcontact-fluorescence imaging system with temperature-controlled,humidity-controlled motorized may be used to monitor muscle activity,e.g., electrophysiological changes, such as action potentials and/orintracellular calcium transients. An integrated fluid-handling systemmay also be used to apply/exchange fluorophores and test compounds, anda microfluidics chamber may be used for simulated drug delivery. Themicrofluidics chamber simulates microvasculature to mimic the manner inwhich a compound/drug contacts a target MTF comprising, e.g.,cardiomyocytes. For example, an MTF may respond differently to aconcentration gradient or different modes of administration. Asignificant advantage of the devices and systems described herein isthat optical mapping system permits detection of such gradient effects,whereas earlier systems, e.g., single cell patch clamp studies, cannotmeasure gradient effects on a cell population.

Appropriate light source and filter sets may be chosen for each desiredfluorophore based on the wavelength of the excitation light andfluoresced light of the fluorophore. Integration of excitationwavelength-switching or an additional detector permits ratiometriccalcium imaging. For this purpose, exemplary fluorophores include Fura-2and Indo-1 or Fluo-3 and Fura Red. For example, excitation and emissionfilters at 515±5 and >695 nm, respectively, are used to measure actionpotentials with di-4-ANEPPS, and excitation and emission filters at365±25 and 485±5 nm, respectively, are used to measure calciumtransients with Indo-1. Automated software may be used and customizedfor data acquisition and data analysis.

Advantages of the optical mapping system include non-invasiveness (nodamage is inflicted to the cell membrane), recorded signals arereal-time action potentials and/or calcium transients in contrast toderivatives of action potentials like extracellular recordings or slowlychanging intracellular ionic concentrations or membrane potential likethe FLIPR system.

For high-throughput optical mapping, analysis may be carried out usingtwo different imaging approaches. For Contact Fluorescence Mapping, amicroscope is not required. Fiber optic cables contact the bottom of aculture plate or wells of a multi-well plate containing MTFs. The plateor wells of the plate are then mapped based on the detectedfluorescence. To screen compounds, test compounds are added to eachindividual well of a multi-well plate, and each bundle of fiber opticcables collects data from each different well providing data pertainingto MTF response to the test compound.

In another embodiment, an inverted microscope may be used to map eachwell individually. Cells of an MTF are contacted with, e.g., achromophore, a fluorophor, or a bioluminescent material, and themicroscope objective is moved from well to well to measure muscleactivities or functions, e.g., electrophysiological changes. Forexample, the response of the MTF to each test compound is monitored foralterations in cardiac excitation, e.g., to identify drugs that induceor do not cause cardiac arrhythmia. Each of the approaches providessignificant advantages (e.g., speed, efficiency, no or minimal usercontact with the MTF, reduced user skill required, ability to observeand measure cell-cell interactions, ability to map action potentialpropagation and conduction velocity, and ability to observe and measurefibrillation and arrhythmia)) compared to previous assays used tomeasure electrophysiological changes (e.g., patch clamp assay in which asingle cell is patch clamped).

These systems are well suited to screen test compounds for, for example,cardiac safety. For example, FDA Guideline S7B addresses “Safetypharmacology studies for assessing the potential for delayed ventricularrepolarization by human pharmaceuticals”. The devices andhigh-throughput in vitro assays described herein allow theidentification of cardiac safety risks much earlier in the drugdiscovery process. The devices and methods of the invention are alsouseful for anti-arrhythmic and/or ion channel-targeted drug discovery.

II. Applications of the Devices of the Invention

The devices of the invention are useful for, among other things,measuring muscle activities or functions, investigating muscledevelopmental biology and disease pathology, as well as in drugdiscovery and toxicity testing.

Accordingly, the present invention also provides methods for identifyinga compound that modulates a contractile function. The methods includeproviding a plurality of muscle thin films; contacting a plurality ofthe muscle thin films with a test compound; and determining the effectof the test compound on a contractile function in the presence andabsence of the test compound, wherein a modulation of the contractilefunction in the presence of the test compound as compared to thecontractile function in the absence of the test compound indicates thatthe test compound modulates a contractile function, thereby identifyinga compound that modulates a contractile function.

In another aspect, the present invention also provides methods foridentifying a compound useful for treating or preventing a muscledisease. The methods include providing a plurality of muscle thin films;contacting a plurality of the muscle thin films with a test compound;and determining the effect of the test compound on a contractilefunction in the presence and absence of the test compound, wherein amodulation of the contractile function in the presence of the testcompound as compared to the contractile function in the absence of thetest compound indicates that the test compound modulates a contractilefunction, thereby identifying a compound useful for treating orpreventing a muscle disease.

The methods of the invention generally comprise determining the effectof a test compound on an MTF as a whole, however, the methods of theinvention may comprise further evaluating the effect of a test compoundon an individual cell type(s) of the MTF.

As used herein, the various forms of the term “modulate” are intended toinclude stimulation (e.g., increasing or upregulating a particularresponse or activity) and inhibition (e.g., decreasing or downregulatinga particular response or activity).

As used herein, the term “contacting” (e.g., contacting a plurality ofMTFs with a test compound) is intended to include any form ofinteraction (e.g., direct or indirect interaction) of a test compoundand an MTF or a plurality of MTFs. The term contacting includesincubating a compound and an MTF or plurality of MTFs together (e.g.,adding the test compound to an MTF or plurality of MTFs in culture).

Test compounds, may be any agents including chemical agents (such astoxins), small molecules, pharmaceuticals, peptides, proteins (such asantibodies, cytokines, enzymes, and the like), nanoparticles, andnucleic acids, including gene medicines and introduced genes, which mayencode therapeutic agents, such as proteins, antisense agents (i.e.,nucleic acids comprising a sequence complementary to a target RNAexpressed in a target cell type, such as RNAi or siRNA), ribozymes, andthe like.

The test compound may be added to an MTF by any suitable means. Forexample, the test compound may be added drop-wise onto the surface of adevice of the invention and allowed to diffuse into or otherwise enterthe device, or it can be added to the nutrient medium and allowed todiffuse through the medium. In the embodiment where the device of theinvention comprises a multi-well plate, each of the culture wells may becontacted with a different test compound or the same test compound. Inone embodiment, the screening platform includes a microfluidics handlingsystem to deliver a test compound and simulate exposure of themicrovasculature to drug delivery. In one embodiment, a solutioncomprising the test compound may also comprise fluorescent particles,and a muscle cell function may be monitored using Particle ImageVelocimetry (PIV).

Numerous physiologically relevant parameters, e.g., muscle activities,e.g., biomechanical and electrophysiological activities, can beevaluated using the methods and devices of the invention. For example,in one embodiment, the devices of the present invention can be used incontractility assays for contractile cells, such as muscular cells ortissues, such as chemically and/or electrically stimulated contractionof vascular, airway or gut smooth muscle, cardiac muscle, vascularendothelial tissue, or skeletal muscle. In addition, the differentialcontractility of different muscle cell types to the same stimulus (e.g.,pharmacological and/or electrical) can be studied.

In another embodiment, the devices of the present invention can be usedfor measurements of solid stress due to osmotic swelling of cells. Forexample, as the cells swell the MTF will bend and as a result, volumechanges, force and points of rupture due to cell swelling can bemeasured.

In another embodiment, the devices of the present invention can be usedfor pre-stress or residual stress measurements in cells. For example,vascular smooth muscle cell remodeling due to long term contraction inthe presence of endothelin-1 can be studied.

Further still, the devices of the present invention can be used to studythe loss of rigidity in tissue structure after traumatic injury, e.g.,traumatic brain injury. Traumatic stress can be applied to vascularsmooth muscle thin films as a model of vasospasm. These devices can beused to determine what forces are necessary to cause vascular smoothmuscle to enter a hyper-contracted state. These devices can also be usedto test drugs suitable for minimizing vasospasm response or improvingpost-injury response and returning vascular smooth muscle contractilityto normal levels more rapidly.

In other embodiments, the devices of the present invention can be usedto study biomechanical responses to paracrine released factors (e.g.,vascular smooth muscle dilation due to release of nitric oxide fromvascular endothelial cells, or cardiac myocyte dilation due to releaseof nitric oxide).

In other embodiments, the devices of the invention can be used toevaluate the effects of a test compound on an electrophysiologicalparameter, e.g., an electrophysiological profile comprising a voltageparameter selected from the group consisting of action potential, actionpotential morphology, action potential duration (APD), conductionvelocity (CV), refractory period, wavelength, restitution, bradycardia,tachycardia, reentrant arrhythmia, and/or a calcium flux parameter,e.g., intracellular calcium transient, transient amplitude, rise time(contraction), decay time (relaxation), total area under the transient(force), restitution, focal and spontaneous calcium release, and wavepropagation velocity. For example, a decrease in a voltage or calciumflux parameter of an MTF comprising cardiomyocytes upon contacting theMTF with a test compound, would be an indication that the test compoundis cardiotoxic.

In yet another embodiment, the devices of the present invention can beused in pharmacological assays for measuring the effect of a testcompound on the stress state of a tissue. For example, the assays mayinvolve determining the effect of a drug on tissue stress and structuralremodeling of the MTF. In addition, the assays may involve determiningthe effect of a drug on cytoskeletal structure (e.g., sarcomerealignment) and, thus, the contractility of the MTF.

In still other embodiments, the devices of the present invention can beused to measure the influence of biomaterials on a biomechanicalresponse. For example, differential contraction of vascular smoothmuscle remodeling due to variation in material properties (e.g.,stiffness, surface topography, surface chemistry or geometricpatterning) of polymeric thin films can be studied.

In further embodiments, the devices of the present invention can be usedto study functional differentiation of stem cells (e.g., pluripotentstem cells, multipotent stem cells, induced pluripotent stem cells, andprogenitor cells of embryonic, fetal, neonatal, juvenile and adultorigin) into contractile phenotypes. For example, undifferentiatedcells, e.g., stem cells, are coated on the thin films anddifferentiation into a contractile phenotype is observed by thin filmbending. Differentiation into an anisotropic tissue may also be observedby quantifying the degree of alignment of sarcomeres and/or quantifyingthe orientational order parameter (OOP). Differentiation can be observedas a function of: co-culture (e.g., co-culture with differentiatedcells), paracrine signaling, pharmacology, electrical stimulation,magnetic stimulation, thermal fluctuation, transfection with specificgenes, chemical and/or biomechanical perturbation (e.g., cyclic and/orstatic strains).

In one embodiment a biomechanical perturbation is stretching of, e.g.,the flexible polymer layer during tissue formation. In one embodiment,the stretching is cyclic stretching. In another embodiment, thestretching is sustained stretching.

In one embodiment, the flexible polymer layer is stretched at anappropriate time after cell seeding that is based on the type(s) ofcells seeded. In one embodiment, the flexible polymer layer is stretchedat about minutes, hours, or days after cell seeding onto a patternedflexible polymer layer. In one embodiment, the flexible polymer layer isstretched at about 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, or about 3.0 hours after cell seeding onto apatterned flexible polymer layer.

In one embodiment, the flexible polymer layer is patternedisotropically. Stretching, therefore, results in the formation ofanisotropic tissue, the anisotropy of which is in the direction of thestretch.

In another embodiment, the flexible polymer layer is patternedanistropically and stretching enhances the anisotropy of the tissueformed.

In one embodiment, the flexible polymer layer is stretched using about a0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10.0 Hertz (Hz) cyclicstretch. In one embodiment, the flexible polymer layer is stretchedusing about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5,15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or about20.0% strength sustained stretch.

In another embodiment, the devices of the invention may be used todetermine the toxicity of a test compound by evaluating, e.g., theeffect of the compound on an electrophysiological response of an MTF.For example, opening of calcium channels results in influx of calciumions into the cell, which plays an important role inexcitation-contraction coupling in cardiac and skeletal muscle fibers.The reversal potential for calcium is positive, so calcium current isalmost always inward, resulting in an action potential plateau in manyexcitable cells. These channels are the target of therapeuticintervention, e.g., calcium channel blocker sub-type ofanti-hypertensive drugs. Candidate drugs may be tested in theelectrophysiological characterization assays described herein toidentify those compounds that may potentially cause adverse clinicaleffects, e.g., unacceptable changes in cardiac excitation, that may leadto arrhythmia.

For example, unacceptable changes in cardiac excitation that may lead toarrhythmia include, e.g., blockage of ion channel requisite for normalaction potential conduction, e.g., a drug that blocks Na⁺ channel wouldblock the action potential and no upstroke would be visible; a drug thatblocks Ca²⁺ channels would prolong repolarization and increase therefractory period; blockage of K⁺ channels would block rapidrepolarization, and, thus, would be dominated by slower Ca²⁺ channelmediated repolarization.

In addition, metabolic changes may be assessed to determine whether atest compound is toxic by determining, e.g., whether contacting with atest compound results in a decrease in metabolic activity and/or celldeath. For example, detection of metabolic changes may be measured usinga variety of detectable label systems such as fluormetric/chrmogenicdetection or detection of bioluminescence using, e.g., AlamarBluefluorescent/chromogenic determination of REDOX activity (Invitrogen),REDOX indicator changes from oxidized (non-fluorescent, blue) state toreduced state (fluorescent, red) in metabolically active cells; VybrantMTT chromogenic determination of metabolic activity (Invitrogen), watersoluble MTT reduced to insoluble formazan in metabolically active cells;and Cyquant NF fluorescent measurement of cellular DNA content(Invitrogen), fluorescent DNA dye enters cell with assistance frompermeation agent and binds nuclear chromatin. For bioluminescent assays,the following exemplary reagents is used: Cell-Titer Gloluciferase-based ATP measurement (Promega), a thermally stable fireflyluciferase glows in the presence of soluble ATP released frommetabolically active cells.

The devices of the invention are also useful for evaluating the effectsof particular delivery vehicles for therapeutic agents e.g., to comparethe effects of the same agent administered via different deliverysystems, or simply to assess whether a delivery vehicle itself (e.g., aviral vector or a liposome) is capable of affecting the biologicalactivity of the MTF. These delivery vehicles may be of any form, fromconventional pharmaceutical formulations, to gene delivery vehicles. Forexample, the devices of the invention may be used to compare thetherapeutic effect of the same agent administered by two or moredifferent delivery systems (e.g., a depot formulation and a controlledrelease formulation). The devices and methods of the invention may alsobe used to investigate whether a particular vehicle may have effects ofitself on the tissue. As the use of gene-based therapeutics increases,the safety issues associated with the various possible delivery systemsbecome increasingly important. Thus, the devices of the presentinvention may be used to investigate the properties of delivery systemsfor nucleic acid therapeutics, such as naked DNA or RNA, viral vectors(e.g., retroviral or adenoviral vectors), liposomes and the like. Thus,the test compound may be a delivery vehicle of any appropriate type withor without any associated therapeutic agent.

Furthermore, the devices of the present invention are a suitable invitro model for evaluation of test compounds for therapeutic activitywith respect to, e.g., a muscular and/or neuromuscular disease ordisorder. For example, the devices of the present invention (e.g.,comprising muscle cells) may be contacted with a candidate compound by,e.g., diffusion of the test compound added drop-wise on the surface ofan MTF, diffusion of a test compound through the culture medium, orimmersion in a bath of media containing the test compound, and theeffect of the test compound on muscle activity (e.g., a biomechanicaland/or electrophysiological activity) may measured as described herein,as compared to an appropriate control, e.g., an untreated MTF.Alternatively, a device of the invention may be bathed in a mediumcontaining a candidate compound, and then the cells are washed, prior tomeasuring a muscle activity (e.g., a biomechanical and/orelectrophysiological activity) as described herein. Any alteration to anactivity determined using the device in the presence of the test agent(as compared to the same activity using the device in the absence of thetest compound) is an indication that the test compound may be useful fortreating or preventing a muscle disease, e.g., a neuromuscular disease.

For use in the methods of the invention, the cells seeded onto the MTFmay be normal muscle cells (cardiac, smooth, or skeletal muscle cells),abnormal muscle cells (e.g., those derived from a diseased tissue, orthose that are physically or genetically altered to achieve a abnormalor pathological phenotype or function), normal or diseased muscle cellsderived from embryonic stem cells or induced pluripotent stem cells, ornormal cells that are seeded/printed onto the film in an abnormal oraberrant configuration. In some cases, both muscle cells and neuronalcells are present on the film.

Evaluation of muscle activity includes determining the degree ofcontraction, i.e., the degree of curvature or bend of the muscular film,and the rate or frequency of contraction/rate of relaxation compared toa normal control or control film in the absence of the test compound. Anincrease in the degree of contraction or rate of contraction indicatesthat the compound is useful in treatment or amelioration of pathologiesassociated with myopathies such as muscle weakness or muscular wasting.Such a profile also indicates that the test compound is useful as avasocontractor. A decrease in the degree of contraction or rate ofcontraction is an indication that the compound is useful as avasodilator and as a therapeutic agent for muscle or neuromusculardisorders characterized by excessive contraction or muscle thickeningthat impairs contractile function.

Compounds evaluated in this manner are useful in treatment oramelioration of the symptoms of muscular and neuromuscular pathologiessuch as those described below. Muscular Dystrophies include DuchenneMuscular Dystrophy (DMD) (also known as Pseudohypertrophic), BeckerMuscular Dystrophy (BMD), Emery-Dreifuss Muscular Dystrophy (EDMD),Limb-Girdle Muscular Dystrophy (LGMD), Facioscapulohumeral MuscularDystrophy (FSH or FSHD) (Also known as Landouzy-Dejerine), MyotonicDystrophy (MMD) (Also known as Steinert's Disease), OculopharyngealMuscular Dystrophy (OPMD), Distal Muscular Dystrophy (DD), andCongenital Muscular Dystrophy (CMD). Motor Neuron Diseases includeAmyotrophic Lateral Sclerosis (ALS) (Also known as Lou Gehrig'sDisease), Infantile Progressive Spinal Muscular Atrophy (SMA, SMA1 orWH) (also known as SMA Type 1, Werdnig-Hoffman), Intermediate SpinalMuscular Atrophy (SMA or SMA2) (also known as SMA Type 2), JuvenileSpinal Muscular Atrophy (SMA, SMA3 or KW) (also known as SMA Type 3,Kugelberg-Welander), Spinal Bulbar Muscular Atrophy (SBMA) (also knownas Kennedy's Disease and X-Linked SBMA), Adult Spinal Muscular Atrophy(SMA). Inflammatory Myopathies include Dermatomyositis (PM/DM),Polymyositis (PM/DM), Inclusion Body Myositis (IBM). Neuromuscularjunction pathologies include Myasthenia Gravis (MG), Lambert-EatonSyndrome (LES), and Congenital Myasthenic Syndrome (CMS). Myopathies dueto endocrine abnormalities include Hyperthyroid Myopathy (HYPTM), andHypothyroid Myopathy (HYPOTM). Diseases of peripheral nerves includeCharcot-Marie-Tooth Disease (CMT) (Also known as Hereditary Motor andSensory Neuropathy (HMSN) or Peroneal Muscular Atrophy (PMA)),Dejerine-Sottas Disease (DS) (Also known as CMT Type 3 or ProgressiveHypertrophic Interstitial Neuropathy), and Friedreich's Ataxia (FA).Other Myopathies include Myotonia Congenita (MC) (Two forms: Thomsen'sand Becker's Disease), Paramyotonia Congenita (PC), Central Core Disease(CCD), Nemaline Myopathy (NM), Myotubular Myopathy (MTM or MM), PeriodicParalysis (PP) (Two forms: Hypokalemic-HYPOP- and Hyperkalemic-HYPP) aswell as myopathies associated with HIV/AIDS.

The methods and devices of the present invention are also useful foridentifying therapeutic agents suitable for treating or ameliorating thesymptoms of metabolic muscle disorders such as Phosphorylase Deficiency(MPD or PYGM) (Also known as McArdle's Disease), Acid Maltase Deficiency(AMD) (Also known as Pompe's Disease), Phosphofructokinase Deficiency(PFKM) (Also known as Tarui's Disease), Debrancher Enzyme Deficiency(DBD) (Also known as Cori's or Forbes' Disease), Mitochondrial Myopathy(MITO), Carnitine Deficiency (CD), Carnitine Palmityl TransferaseDeficiency (CPT), Phosphoglycerate Kinase Deficiency (PGK),Phosphoglycerate Mutase Deficiency (PGAM or PGAMM), LactateDehydrogenase Deficiency (LDHA), and Myoadenylate Deaminase Deficiency(MAD).

In addition to the disorders listed above, the screening methodsdescribed herein are useful for identifying agents suitable for reducingvasospasms, heart arrhythmias, and cardiomyopathies.

Vasodilators identified as described above are used to reducehypertension and compromised muscular function associated withatherosclerotic plaques. Smooth muscle cells associated withatherosclerotic plaques are characterized by an altered cell shape andaberrant contractile function. Such cells are used to populate a thinfilm, exposed to candidate compounds as described above, and muscularfunction evaluated as described above. Those agents that improve cellshape and function are useful for treating or reducing the symptoms ofsuch disorders.

Smooth muscle cells and/or striated muscle cells line a number of lumenstructures in the body, such as uterine tissues, airways,gastrointestinal tissues (e.g., esophagus, intestines) and urinarytissues, e.g., bladder. The function of smooth muscle cells on thinfilms in the presence and absence of a candidate compound may beevaluated as described above to identify agents that increase ordecrease the degree or rate of muscle contraction to treat or reduce thesymptoms associated with a pathological degree or rate of contraction.For example, such agents are used to treat gastrointestinal motilitydisorders, e.g., irritable bowel syndrome, esophageal spasms, achalasia,Hirschsprung's disease, or chronic intestinal pseudo-obstruction.

The present invention is next described by means of the followingexamples. However, the use of these and other examples anywhere in thespecification is illustrative only, and in no way limits the scope andmeaning of the invention or of any exemplified form. The invention isnot limited to any particular preferred embodiments described herein.Many modifications and variations of the invention may be apparent tothose skilled in the art and can be made without departing from itsspirit and scope. The contents of all references, patents and publishedpatent applications cited throughout this application, including thefigures, are incorporated herein by reference.

EXAMPLES Example 1 Muscular Thin Film Device and Use Thereof forDetermining a Contractile Function

A. Substrate Fabrication

Polydimethylsiloxane (PDMS) thin film substrates were fabricated via amulti-step spin coating process. Poly(N-isopropylacrylamide) (PIPAAm)(Polysciences, Inc.) was dissolved at 10 wt % in 99.4% 1-butanol (w/v)and spun coat onto the glass cover slips. Sylgard 184 (Dow Corning)polydimethylsiloxane (PDMS) elastomer was mixed at a 10:1 base to curingagent ratio, doped with 0.1% by volume 0.2 μm Fluorospheres(Invitrogen), and spin coated on top of the PIPAAm coated glass coverslip. Polydimethylsiloxane-coated cover slips were then cured.

B. ECM Patterning

The polydimethylsiloxane thin films were coated with either an isotropicor anisotropic layer of extracellular matrix (ECM) (e.g. fibronectin,laminin, collagen I). In each case, immediately prior to ECM treatment,the polydimethylsiloxane-coated cover slips were UV ozone treated for 8minutes to sterilize the surface and increase hydrophilicity. Allsubsequent processing was performed in a biohood under sterileconditions.

ECM patterning was performed using microcontact printing (μCP). Thebasic μCP technique is well established and allows the rapid patterningof biomolecules on a variety of planar substrates usingpolydimethylsiloxane stamps. The variation employed here used apolydimethylsiloxane stamp to pattern ECM proteins on thepolydimethylsiloxane coated glass cover slips to form anisotropic 2Dmyocardium. ECM proteins were transferred from the stamp to thepolydimethylsiloxane thin film by making conformal contact for 1 minute.

C. 1. Vascular Smooth Muscle Seeding and Culture

Human umbilical artery vascular smooth muscle was seeded on thin filmsat 300 cells/mm² in complete M199 medium (M199 supplemented with 10%fetal bovine serum (FBS), penicillin, streptomycin, L-glutamine, glucoseand vitamin B12). The tissue was cultured in complete M199 for 48 hours,with a single media change at 24 hours. After 48 hours, the cells wereserum starved for an additional 48 hours (M199 supplemented withpenicillin, streptomycin, L-glutamine, glucose and vitamin B12, but noFBS).

2. Neonatal Rat Ventricular Myocytes Seeding and Culture

Neonatal rat ventricular myocytes were isolated from 2-day old neonatalSprague-Dawley rats based on published methods. Briefly, ventricles wereextracted and homogenized by washing in Hanks balanced salt solutionfollowed by digestion with trypsin and collagenase with agitationovernight at 4° C. Cells were re-suspended in M199 culture mediumsupplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS),10 mM HEPES, 3.5 g/L glucose, 2 mM L-glutamine, 2 mg/L vitamin B-12, and50 U/ml penicillin and seeded on anisotropically patterned FN at adensity of 1 million cells per cover slip. Samples were incubated understandard conditions at 37° C. and 5% CO2. Media was exchanged withmaintenance media (2% FBS) every 48 h until use. The MTFs were culturedfor a period of 4-6 days and then used in the contractility assay.

D. Thin Film Release

Polydimethylsiloxane films were transferred to a petri dish of Tyrode'ssolution at 37° C. The Petri dish was placed on a stereomicroscope withdarkfield illumination and cut into rectangles, parallel with tissueorientation, using a razor blade. Once the Tyrode's solution cools below35° C., the PIPAAm layer transitions from a hydrophobic state to ahydrophilic state and begins to dissolve. Once the PIPAAm dissolves,films are pulled free of the coverslip with tweezers.

E. Experimental Testing Parameters (Tyrodes, Pacing, Video Recording)

Muscle thin films were transferred to fresh Tyrode's solution in thetesting dish (FIG. 1), which is maintained at 37° C. by a peltierheating system. Films were adhered to Teflon coated posts (FIG. 3) bymoving them into close contact using tweezers. The films were imagedusing a stereomicroscope outfitted with fluorescent imaging capability.Both brightfield and fluorescent images were captured every thirtyseconds. In the experiment shown in FIG. 4, four films were treated with50 nM endothelin-1 followed by 100 μM HA-1077 (fasudil).

F. Image and Stress Analysis

Quantification and analysis of thin film motion was performed usingImageJ (NIH) and MATLAB software. Thresholded fluorescent images (FIG. 4B) were fit to circles whose radii of curvature could be measured (FIG.4C). Using elasticity theory, the contraction stress necessary to inducethe measured changes in curvature were calculated (FIG. 4D).

Results

Muscular thin films were engineered using both cardiomyocytes (cMTFs)and vascular smooth muscle cells (vMTFs). MTFs were constructed byseeding dissociated muscle cells on a multilayer polymer substrate.PIPAAm was spin coated onto a glass coverslip and PDMS is spin coatedonto of the PIPAAm layer. The cells were seeded on ECM proteinsmicropatterned onto the PDMS layer. When the media temperature waslowered below 35° C., the PIPAAm dissolves and the MTF was released andfree floating. The radius of curvature of the resulting bilaminatestructure is indicative of the stress in the cell layer.

In order to guide alignment of the cells, lines of ECM proteins (FN orLAM) were microcontact printed onto the PDMS. Atomic Force Microscopy(AFM) surface scanning revealed that this ECM pattern is approximately10-20 nm in thickness. When cells were cultured on the patterned ECM,they spontaneous organize into a contiguous tissue that is aligned withthe patterned matrix lines.

In these cells, the long axis of the cell and the nuclear eccentricityparallel the underlying matrix pattern. Fluorescent staining of thefixed tissues revealed that the actin aligns with the underlying matrixin the vascular smooth muscle. In cardiac muscle the sarcomeric Z-linesare perpendicular to the matrix. In both cases, the cytoskeletalarchitecture indicates that the primary axis of contraction is in thelongitudinal direction.

Cardiac ventricular MTFs (cMTFs) were used to measure systoliccontraction stress and contractile wave speed in engineered myocardialtissues. The cMTFs were engineered as anisotropic, with uniaxialcellular alignment. Imaging of the Z-disks by immunostaining sarcomericα-actinin confirmed that alignment of the myofibrils was alsoanisotropic. The cMTFs were mounted in a bath with a PDMS clamp tofirmly hold the cMTF in place during rapid movement and parallelplatinum wire electrodes to field stimulate muscle contraction. Duringdiastole, the cMTF may have a baseline curvature due to resting tensionin the tissue construct. During contraction, the radius of curvaturedecreases dramatically, due to cardiomyocytegenerated stress of 13.9kPa. In this set of experiments, three cMTFs built from the ventricularmyocytes harvested from two different rat pup litters produced a meanpeak systolic stress of 9.2±3.5 kPa.

The cMTF goes through substantial bending and deformation duringcontraction, however, because the cMTF is a thin beam, the actualshortening of the cardiomyocytes is <1%, i.e. contraction is isometric.Knowing the stress generated by the cMTF and the elastic modulus of thecardiomyocytes (E=˜30 kPa), the unconstrained shortening was estimatedas 25% at peak systole. The accurate measurement of the cell and PDMSlayer thicknesses are critical for calculating the contraction stressand unconstrained shortening. The elastic modulus of the cell layer,however, has little affect on the calculated stress but does have asignificant effect is on the calculation of λa, so calculatingunconstrained shortening is strongly dependent on accurate cell modulusmeasurements.

The cMTF system is able to provide an estimate of the contractile wavevelocity based on the mechanical deformation of the cMTFs. Tracking themechanicalwave requires that contraction in the cMTF initiates at oneend and propagates to the other. Spontaneous contraction of cMTFs ofteninitiated at the free end and propagated to the base. Tracking theinitial position and propagation of maximum curvature along the cMTFfrom the initiation of contraction until peak systole (uniformcurvature) enabled estimation of contractile wave propagation. In theexample shown here, the contractilewave speed was 1.875 cm/s, comparableto the velocity of the mechanical wave reported using phase imagingtechniques.

Vascular MTFs (vMTFs) were used to demonstrate the potential of thismethod as a pharmaceutical screening assay. Human umbilical artery VSMCswere engineered as anisotropic monolayers aligned parallel to the longaxis of the MTF. The vMTFs were adhered to PTFE coated posts viahydrophobic interaction with the PDMS. This arrangement allowed multiplevMTFs to be viewed concurrently. Here, eight vMTFs were tested usingthis assay. The fluorosphere doped PDMS could be viewed usingfluorescent stereomicroscopy. In this arrangement, the films are easilyapproximated as circular arcs.

The vMTFs were treated with the endothelium-produced vasoconstrictorendothelin-1 followed by the rho-kinase inhibitor HA-1077, in order tocalculate all of the relevant stress states of arterial muscle. ThevMTFs had an initial stable baseline curvature indicating that the cellsgenerated a basal stress, defined as the sum of the passive residualstress and the basal contractile tone, of 17.1±1.7 kPa. This valuerepresents the resting tension of the tissue. At time 0, the vMTFs werestimulated with 50 nM ET-1, inducing contraction, which caused adecrease in their radii of curvature as the cell generated stressincreased by 5.06±0.75 kPa. Treatment with 100 mM HA-1077, a rho-kinaseinhibitor, caused a rapid increase in radius of curvature, due toinhibition of contraction (FIG. 6G), and resulted in a stress of 3.1±0.8kPa. The HA-1077 dosage is sufficient to inhibit all myosin light chainphosphorylation, so this value represents the residual stress, or thestress generated by the cytoskeletal elements not involved in thecontractile apparatus, but which remains after all other loads areremoved. By comparing the residual stress following HA-1077 treatment tothe pre ET-1 treated tissue, it can be can determined that the vMTFs hada basal contractile tone of 13.1±2.1 kPa. This protocol demonstratesthat the vMTFs are able to mimic well documented native vascularbehavior and implies that this assay could be used to test the effectsof pharmaceutical agents on vascular contractility.

The measured peak systolic stress and constrained shortening of themuscle thin films (MTF) fabricated using neonatal rat ventricularmyocytes as described above, were comparable to isometric measurement ofisolated papillary muscle. Thus, this MTF system recapitulates both theanisotropic alignment of normal cardiac muscle and physiologicallyrelevant, systolic stress levels.

A unique aspect of the MTF contractility assay is the capability totrack local changes in radius of curvature along its length during thecardiac cycle. Further, dyssynchronous contraction when the mechanicalwave fails to propagate, or initiates at multiple locations at the sametime, resulting in a fluttering cMTF with no discernable deflection canbe detected. Thus, a broad range of qualitative and quantitative datacan be extracted from the assay by proper frame by frame analysis of thedeformation.

The vascular smooth muscle cell MTFs were used to mimic the lamellae ofthe arterial tunica media. The vMTF assay confirms the presence offunctional ET-1 receptors in the engineered smooth muscle and is able toaccurately quantify the magnitude of induced contraction. Concurrentmonitoring of eight vMTFs contracting and relaxing demonstrates thatthis technique produces engineered smooth muscle that repeatabilityresponds to pharmacologic stimulation at physiologic stress levels.Moreover, this assay demonstrates that MTFs can provide a method forstudying diseases and potential therapeutic interventions, with thepotential to significantly scale up the throughput. As an earlyscreening method, this high-fidelity, in vitro contractility assay couldbe used to directly test the effect of drugs on contractility andpotentially decrease the high failure rate of cardiovascular drugs, ascurrently, novel molecules reaching Phase 1 clinical trials forcardiovascular drugs have a completion rate of less than 20%.

The methods and devices described above and below for the preparation ofmucle thin films (MTFs) permit the preparation of a more relevant invitro model of engineered tissue in that the engineered tissue displaysone or more properties of mature tissues, e.g., matureelectrophysiology, such as mature action potential morphology, matureion channel expression, and mature contractility, rather than theimmature properties displayed by tissues/cells cultured using previouslydescribed methods. Also see, e.g., WO 2008/051265.

Example 2 Cardiac Myocytes and Muscular Thin Film High Content, EnhancedThroughput Device and Use Thereof for Determining a Contractile Function

A. Substrate Fabrication

Polydimethylsiloxane (PDMS) thin film substrates were fabricated via amulti-step spin coating process. Poly(N-isopropylacrylamide) (PIPAAm)(Polysciences, Inc.) was dissolved at 10 wt % in 99.4% 1-butanol (w/v)and the PIPAAm is deposited in the mid-section of the cover-slip (asshown in FIG. 7A(i)). Sylgard 184 (Dow Corning) polydimethylsiloxane(PDMS) elastomer was mixed at a 10:1 base to curing agent ratio and spuncoat on top of the PIPAAm coated glass cover slip (FIG. 7A(ii)).Polydimethylsiloxane-coated cover slips were then cured.

Substrates suitable for the horizontal muscle thin films were alsofabricated using Sylgard 184 (Dow Corning) polydimethylsiloxane (PDMS)elastomer mixed at a 10:1 base to curing agent ratio, doped with 0.1% byvolume 0.2 μm Fluorospheres (Invitrogen), and spun coated on top of thePIPAAm coated glass cover slip which were then cured.

B. Fibronectin Anisotropic Patterning

The PDMS thin films were coated with an anisotropic layer of fibronectin(FN). In each case, immediately prior to fibronectin treatment, thePDMS-coated cover slips were UV ozone treated for 8 minutes to sterilizethe surface and increase hydrophilicity. All subsequent processing wasperformed in a biohood under sterile conditions.

Anisotropic patterning of fibronectin was performed using microcontactprinting (μCP). The basic μCP technique is well established and allowsthe rapid patterning of biomolecules on a variety of planar substratesusing PDMS stamps. The variation employed here used apolydimethylsiloxane stamp to pattern fibronectin on thepolydimethylsiloxane coated glass cover slips to form anisotropic 2Dmyocardium. Fibronectin (50 μg/mL fibronectin in sterile deionized (DI)water) was transferred from the stamp to the polydimethylsiloxane thinfilm by making conformal contact for 1 minute. The stamp was position insuch a way that the pattern is perpendicular to the PIPAAm deposit (FIG.7A(iv)). The films were then incubated for 15 minutes in lowconcentration fibronectin (2.550 μg/mL fibronectin in sterile DI water).Following incubation, excess fibronectin was removed by washing 3 timeswith a sterile phosphate buffer solution (PBS) and then left in PBSuntil seeding.

C. Cardiomyocyte Seeding and Culture

Neonatal rat ventricular myocytes were isolated from 2-day old neonatalSprague-Dawley rats based on well known methods. Cells were diluted to aconcentration of ˜350,000 per mL in seeding media (SM) (M199 mediasupplemented with 10% FBS), and 3 mL was seeded on each cover slip.After 24 hours incubation, the cover slips were washed 3 times withphosphate buffered saline (PBS) to remove non-adherent cells andrecovered with SM. After an additional 24 hours, the media was exchangedwith maintenance media (MM) [M199 media supplemented with 2% fetalbovine serum (FBS)] to minimize growth of fibroblasts inevitably presentin the primary harvest cardiomyocyte population.

D. Releasing the Films for a Contractility Study

MTFs were released from the cover slip once the cells have formed theappropriate 2D microstructure. The MTFs were either cut out by hand, cutout using a robotic system, pre-cut prior to cell incubation. In onespecific example of, e.g., horizontal MTF production, the middle sectionwas cut out, so that only eight rectangles remained in the area that hadPIPAAm (see FIG. 7A(vi) for example of the cutout shape). Once theTyrode's solution cools below 35° C., the PIPAAm layer transitions froma hydrophobic state to a hydrophilic state and begins to dissolve. Asthe PIPAAm dissolved the middle section cutout was peeled off thesubstrate with a pair of tweezers. Once the PIPAAm dissolves completely,the contraction of the myocytes pulls the MTF (the remaining rectangles)free from the rigid substrate. In the case of the horizontal MTF,rectangles remain with one end partially fixed to the substrate (FIG.7A(vii)).

E. Experimental Testing Parameters (Tyrodes, Pacing, Video Recording)

Actuation and observation of multiple MTFs was carried out in aphysiologic solution (e.g., normal Tyrodes solution). MTFs wereelectrically paced using parallel platinum wire electrodes spaced ˜1 cmapart and lowered directly into the center of the Petri dish. Anexternal field stimulator (Myopacer, IonOptix) was used to apply a 10-20V, 10 msec duration square wave between the electrodes at pacing ratesfrom 0.5 to 2 Hz.

F. Video and Image Analysis

Quantification and analysis of thin film motion was performed usingImageJ (NIH) (FIG. 7B) and MATLAB software. From the thresholdedfluorescent images the films length was tracked throughout thecontraction and the radius of curvature calculated (FIGS. 7C and 7D).Using elasticity theory, the contraction stress necessary to induce themeasured changes in curvature were calculated.

Example 3 Multi-Well Plate Tissue Contractility Device and Assay

This example describes the fabrication of a multi-well plate withmicropatterned bottom surfaces wherein each well is used in a horizontalmuscular thin film (MTF) assay (see, e.g., FIG. 9A). The micropatternedmulti-well plates are especially amenable to assays using automaticimaging of the MTFs in, e.g., a GE InCellAnalyzer.

The multi-well plate with MTF assay, can be used for biomechanicalmeasurements of myocyte contraction properties. In this device thetissue constructs remain partially fixed to a hard substrate, such asthe glass bottom of the multi-well plate. This invention is designed foruse with polymeric thin films (PCT Publication No. WO 2008/051265 A2),but could be used with any differentially stressed polymer or tissueconstruct. In this assay the thin films bend up from the viewing(horizontal) plane unlike the original thin film assay in which a singlefilm bends in the viewing plane. The number of wells is limited by thesize of the desired films (the films have to be large enough for thecells to constitute a tissue), with a film of at least 1.2 mm×2.4 mm any6, 12, 24, 48, 96 well plates can be used. The assay may be utilized tomeasure biomechanical forces due to a number of stimuli including, butnot limited to, contraction, osmotic swelling, structural remodeling andtissue level pre-stress. It is possible to further automate the assay bymethods that include, but are not limited to, cookie cutter razors thatwould come down into the wells to cut the films, programmable lasersthat cut the films, and automatic aspiration pipettes to aspirate outunwanted film sections. The biomechanical responses due to paracrinesignaling events can also be studied through the addition of aco-culture system, making the device attractive for studyingcell-to-cell drug effects.

In the context of the present experiment a section of glass was cut tomatch the dimensions of the multi-well plate skeleton section of glass(7.5 cm×11 cm) and was covered with a protective film (to preserve theoptical clarity of the substrate during later fabrication steps), bylowering the protective film onto the glass covered with 200 proof ethylalcohol, and removing, e.g., using pressure, the excess ethanol fromunder the film. This process was repeated for the other side of theglass and then islands corresponding to a portion of the wells were cutout of the top protective film. Then, a temperature sensitive polymer,specifically poly(N-isopropylacrylamide) (PIPAAm), was deposited in athin layer onto the open glass islands and the top protective film layerwas peeled off. A biopolymer, specifically polydimethylsiloxane (PDMS),was deposited in a layer, e.g., by spin coating, (˜5-25 μm) on top ofthe whole glass, with the bottom protective film preventingback-splatter of the PDMS onto the glass. The PDMS was allowed tocompletely cure overnight and the bottom protective film was peeled off.A biopolymer, e.g., extracellular matrix protein (ECM), e.g.,fibronectin (FN), was stamped (micro-contact printed) in a pattern ontothe PDMS.

In parallel, a second section of glass was cut to match the dimensionsof the multi-well plate skeleton (7.5 cm×11 cm). The second glasssection was then spin coated with a layer of PDMS, which was immediatelybrought into contact with and lifted off of the bottom of the multi-wellplate skeleton leaving behind a thin layer of PDMS on the bottom of themulti-well plate skeleton. At this point, the ECM patterned PDMS-glassbase was pressed into contact with the PDMS coated bottom of themulti-well plate skeleton (24 well A-Plate GBMP Black Porvair SciencesLtd.), creating a PDMS seal between the wells. To provide additionalliquid load bearing capacity, a sealant was painted around the outerborder, further adhering the glass base to the multi-well plateskeleton. The plate was then placed in a humid warm (37° C.) incubatorovernight to complete the PDMS seal curing while providing the humiditynecessary to maintain proper ECM molecule activity.

Phenol red was placed inside various wells of the plate to confirm thatthe seal between wells did not leak. Following a 48-hour incubation, thephenol red had not spread to adjacent wells demonstrating that the sealsof the wells were intact (see, e.g., FIG. 9B).

Contractile cells, specifically cardiomyocytes, were seeded onto the ECMinside each well. For the multi-well plate, the MTFs inside the filmwith the cells were cut and the unwanted regions peeled off the glass ineach well. As a result a single rectangle of film remained attached tothe glass at one edge only in each well. The dynamics of these tissueconstructs was recorded. The cells in the plate can be fixed andimmuno-stained to study cell structure (see, e.g., FIG. 9C).

Example 4 High-Throughput Multi-Tissue Contractility Device and Assay

A multi-tissue contractility assay can be used, for example, toqualitatively compare contractions of two tissue types, to compare theeffect of one tissue response in proximity to another tissue, or forbiomechanical measurements of myocyte contraction properties.

The substrates for use in a multi-tissue contractility assay are made asdescribed below and herein. In this device, the tissue constructs remainpartially fixed to a rigid substrate, e.g., the glass bottom of themulti-well plate, and bend up from the viewing (horizontal) plane. Therigid substrate is made in such a manner that it can be split after theECM has been patterned into as many parts, at least two, as the numberof tissues. Different types of cells are then cultured on the substrateor different types of micropatterning can be patterned onto thesubstrate, e.g., line patterns, anisotropic monolayers, or isotropicmonolayers. The assay could be utilized to measure biomechanical forcesdue to a number of stimuli including, but not limited to contraction,osmotic swelling, structural remodeling and tissue level pre-stress. Thebiomechanical responses due to paracrine signaling events can also bestudied, making the device attractive for studying cell-to-cell drugeffects.

One benefit of the methods described below is the ability to maintaineven thickness of the sacrificial polymer layer from island to island,thus, yielding more consistent devices.

In the context of the present experiment and as depicted in FIGS. 10 and11, a section of glass (7.5 cm×11 cm) was covered with a protectivefilm, by lowering the protective film onto the glass covered with 200proof ethyl alcohol, and removing, e.g., using pressure, the excessethanol from under the film. This process was repeated for the otherside of the glass and then islands corresponding to the desired size ofassay were cut out of the top film. A temperature sensitive polymer,specifically poly(N-isopropylacrylamide) (PIPAAm), was then deposited asa thin layer onto the open glass islands, then the top protective filmlayer was peeled off. A polymer, specifically polydimethylsiloxane(PDMS), was deposited in a layer (˜5-25 μm) on top of the whole glass,with the bottom protective film preventing back-splatter of the PDMSonto the glass. The PDMS was allowed to completely cure overnight andthe bottom protective film was peeled off. A mask, printed ontransparencies was used to cut the glass into desired shapes. Anextracellular matrix protein, such as (ECM) fibronectin (FN), wasstamped in the pattern depicted in FIGS. 10 and 11 onto the PDMS. Theglass was cut in between the two PIPAAm islands. The two pieces of glasswere treated with different agents (Pluronic F127 blocking and lowconcentration FN background). Contractile cells, such as cardiomyocytes,were seeded onto the ECM. The cells formed lines in the first (Pluronicstreated) tissue, and anisotropic monolayers in the second tissue (lowconcentration FN treated). The cover-slips were then combined and thefilms cut, with the unwanted regions peeled away. As a result a singlerectangle of film remained attached to the glass at one edge only. Thedynamics of these tissue constructs were recorded.

Example 5 Vascular Smooth Muscle Thin Film High Content, EnhancedThroughput Device and Use Thereof for Determining a Contractile Function

A. Substrate Fabrication

A section of glass (7.5 cm×11 cm) was covered with a static vinylprotective film by lowering the protective film onto the glass, andremoving, e.g., using pressure, the all air bubble from under the film.This process was repeated for the other side of the glass and thenislands corresponding to the desired size of assay were cut out of thetop film.

Polydimethylsiloxane (PDMS) thin film substrates were fabricated via amulti-step spin coating process. Poly(N-isopropylacrylamide) (PIPAAm)(Polysciences, Inc.) was dissolved at 10 wt % in 99.4% 1-butanol (w/v)and was then deposited as a thin layer onto the open glass islands, thenthe top protective film layer was peeled off. Sylgard 184 (Dow Corning)polydimethylsiloxane (PDMS) elastomer was mixed at a 10:1 base to curingagent ratio doped with 0.1% by volume 0.2 mm Fluorospheres (Invitrogen),and spun coated on top of the PIPAAm coated glass cover slip which wasthen cured. The next day, the bottom protective film was peeled off. Amask, printed on transparencies was used to cut the glass into desiredshapes.

B. Fibronectin Anisotropic Patterning

The PDMS thin films were coated with an anisotropic layer of fibronectin(FN). In each case, immediately prior to fibronectin treatment, thePDMS-coated cover slips were UV ozone treated for 8 minutes to sterilizethe surface and increase hydrophilicity. All subsequent processing wasperformed in a biohood under sterile conditions.

Anisotropic patterning of fibronectin was performed using microcontactprinting (mCP). The basic mCP technique is well established and allowsthe rapid patterning of biomolecules on a variety of planar substratesusing PDMS stamps. The variation employed here used apolydimethylsiloxane stamp to pattern fibronectin on thepolydimethylsiloxane coated glass cover slips to form anisotropic 2Dmyocardium. Fibronectin (50 μg/mL fibronectin in sterile deionized (DI)water) was transferred from the stamp to the polydimethylsiloxane thinfilm by making conformal contact for 1 minute. The stamp was positionedin such a way that the pattern is perpendicular to the PIPAAm deposit.Following stamping, excess fibronectin was removed by washing 3 timeswith a sterile phosphate buffer solution (PBS) and then left dry untilseeding.

C. Neonatal Rat Ventricular Myocytes Seeding and Culture

Neonatal rat ventricular myocytes were isolated from 2-day old neonatalSprague-Dawley rats based on published methods. Briefly, ventricles wereextracted and homogenized by washing in Hanks balanced salt solutionfollowed by digestion with trypsin and collagenase with agitationovernight at 4° C. Cells were re-suspended in M199 culture mediumsupplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS),10 mM HEPES, 3.5 g/L glucose, 2 mM L-glutamine, 2 mg/L vitamin B-12, and50 U/ml penicillin and seeded on anisotropically patterned FN at adensity of 1 million cells per cover slip. Samples were incubated understandard conditions at 37° C. and 5% CO2. Media was exchanged withmaintenance media (2% FBS) every 48 h until use. The MTFs were culturedfor a period of 4-6 days and then used in the contractility assay.

D. Releasing the Films for a Contractility Study

MTFs were released from the cover slip once the cells have formed theappropriate 2D microstructure. The MTFs were either cut out by hand, cutout using a robotic system, pre-cut prior to cell incubation. In onespecific example of, e.g., horizontal MTF production, the middle sectionwas cut out, so that only six rectangles remained in the area that hadPIPAAm). Once the Tyrode's solution cools below 35° C., the PIPAAm layertransitions from a hydrophobic state to a hydrophilic state and beginsto dissolve. As the PIPAAm dissolved the middle section cutout waspeeled off the substrate with a pair of tweezers. Once the PIPAAmdissolves completely, the contraction of the myocytes pulls the MTF (theremaining rectangles) free from the rigid substrate. In the case of thehorizontal MTF, rectangles remain with one end partially fixed to thesubstrate.

E. Experimental Testing Parameters (Tyrodes, Pacing, Video Recording)

Actuation and observation of multiple MTFs was carried out in aphysiologic solution (e.g., normal Tyrodes solution). The horizontalMTFs (hMTFs) fabricated with vascular smooth muscle cells in thepresence of fluorescent beads were treated with the endothelium-producedvasoconstrictor endothelin-1 (ET-1) followed by the rho-kinase inhibitorHA-1077.

The hMTFs had an initial stable baseline curvature (FIG. 12) indicatingthat the cells generated a basal stress. At time 0, the hMTFs werestimulated with 50 nM ET-1, inducing contraction, which caused adecrease in their radii of curvature (FIG. 12). Treatment with 100 mMHA-1077, a rho-kinase inhibitor, caused a rapid increase in radius ofcurvature, due to inhibition of contraction.

This protocol demonstrates that the hMTFs are able to mimic welldocumented native vascular behavior and implies that this assay could beused to test the effects of pharmaceutical agents on vascularcontractility.

F. Video and Image Analysis

Quantification and analysis of thin film motion was performed usingImageJ (NIH) and MATLAB software. From the thresholded fluorescentimages the length of the projection of the film on the horizontal planewas tracked throughout the contraction and the radius of curvaturecalculated. Using elasticity theory, the contraction stress necessary toinduce the measured changes in curvature were calculated.

EQUIVALENTS

In describing embodiments of the invention, specific terminology is usedfor the sake of clarity. For purposes of description, each specific termis intended to at least include all technical and functional equivalentsthat operate in a similar manner to accomplish a similar purpose.Additionally, in some instances where a particular embodiment of theinvention includes a plurality of system elements or method steps, thoseelements or steps may be replaced with a single element or step;likewise, a single element or step may be replaced with a plurality ofelements or steps that serve the same purpose. Further, where parametersfor various properties are specified herein for embodiments of theinvention, those parameters can be adjusted up or down by 1/20th,1/10th, ⅕th, ⅓rd, ½, etc., or by rounded-off approximations thereof,unless otherwise specified. Moreover, while this invention has beenshown and described with references to particular embodiments thereof,those skilled in the art will understand that various substitutions andalterations in form and details may be made therein without departingfrom the scope of the invention; further still, other aspects, functionsand advantages are also within the scope of the invention. The contentsof all references, including patents and patent applications, citedthroughout this application are hereby incorporated by reference intheir entirety. The appropriate components and methods of thosereferences may be selected for the invention and embodiments thereof.Still further, the components and methods identified in the Backgroundsection are integral to this disclosure and can be used in conjunctionwith or substituted for components and methods described elsewhere inthe disclosure within the scope of the invention.

1. A method for identifying a compound that modulates a contractilefunction, the method comprising providing a plurality of muscle thinfilms; contacting a plurality of said muscle thin films with a testcompound; and determining the effect of the test compound on acontractile function in the presence and absence of the test compound,wherein a modulation of the contractile function in the presence of saidtest compound as compared to the contractile function in the absence ofsaid test compound indicates that said test compound modulates acontractile function, thereby identifying a compound that modulates acontractile function.
 2. A method for identifying a compound useful fortreating or preventing a muscle disease, the method comprising providinga plurality of muscle thin films; contacting a plurality of said musclethin films with a test compound; and determining the effect of the testcompound on a contractile function in the presence and absence of thetest compound, wherein a modulation of the contractile function in thepresence of said test compound as compared to the contractile functionin the absence of said test compound indicates that said test compoundmodulates a contractile function, thereby identifying a compound usefulfor treating or preventing a muscle disease.
 3. The method of claim 1,wherein the contractile function is a biomechanical activity.
 4. Themethod of claim 3, wherein the biomechanical activity is selected fromthe group consisting of contractility, cell stress, cell swelling, andrigidity.
 5. The method of claim 1, wherein the contractile function isan electrophysiological activity.
 6. The method of claim 5, wherein theelectrophysiological activity is a voltage parameter selected from thegroup consisting of action potential, action potential duration (APD),conduction velocity (CV), refractory period, wavelength, restitution,bradycardia, tachycardia, and reentrant arrhythmia.
 7. The method ofclaim 5, wherein said electrophysiological activity is a calcium fluxparameter selected from the group consisting of intracellular calciumtransient, transient amplitude, rise time (contraction), decay time(relaxation), total area under the transient (force), restitution, focaland spontaneous calcium release.
 8. The method of claim 1, furthercomprising applying a stimulus to the plurality of muscle thin films. 9.The method of claim 1, wherein the plurality of muscle thin films areadhered to a solid support structure.
 10. (canceled)
 11. The method ofclaim 1, wherein the plurality of muscle thin films are cultured in thepresence of a fluorophor. 12-18. (canceled)
 19. The method of claim 1,wherein said plurality of muscle thin films comprises cardiomyocytes.20. The method of claim 1, wherein said plurality of muscle thin filmscomprises vascular smooth muscle cells or vascular endothelial cells.21. The method of claim 1, wherein said plurality of muscle thin filmscomprises smooth muscle cells or striated muscle cells.
 22. A device formeasuring a contractile function, the device comprising: a solid supportstructure; a plurality of muscle thin films adhered to said solidsupport structure, wherein said plurality of muscle thin films eachcomprise a flexible polymer layer and a population of isolated cellsseeded on the flexible polymer layer in a predetermined pattern, andwherein said cells form a tissue structure which can perform acontractile function.
 23. The device of claim 22, wherein the pluralityof muscle thin films are adhered to said solid support structuredirectly.
 24. The device of claim 22, wherein the plurality of musclethin films are adhered to said solid support structure indirectly. 25.The device of claim 24, wherein the plurality of muscle thin films areadhered indirectly by a plurality of posts attached to said solidsupport structure.
 26. A device for measuring a contractile function,the device comprising: a solid support structure; a plurality of musclethin films adhered at one end to said solid support structure, whereinthe plurality of muscle thin films comprise a flexible polymer layer anda population of isolated cells seeded on the flexible polymer layer in apredetermined pattern, wherein said cells form a tissue structure whichcan perform a contractile function.
 27. The device of claim 22, furthercomprising a second solid support structure seeded with a secondpopulation of cells.
 28. The device of claim 22, wherein the solidsupport structure is a glass coverslip, a Petri dish, strips of glass,glass slides, or a multi-well plate.
 29. The device of claim 22, whereinthe flexible polymer layer comprises polydimethylsiloxane.
 30. Thedevice of claim 22, wherein the cells are myocytes.
 31. The device ofclaim 30, wherein the myocytes are cardiomyocytes.
 32. The device ofclaim 30, wherein the myocytes are aligned to produce an anisotropictissue.
 33. The device of claim 22, further comprising supplying anengineered surface chemistry to the flexible polymer layer beforeseeding the cells on the flexible polymer layer.
 34. The device of claim33, wherein the engineered surface chemistry comprises an extracellularmatrix protein.
 35. The device of claim 33, wherein the engineeredsurface chemistry is provided in a pattern that includes gaps.
 36. Thedevice of claim 22, wherein the solid support structure comprises aplurality of cell culture wells; an optical signal capture device; andan image processing software to calculate change in an optical signal.37. The device of claim 36, wherein said optical signal capture devicecomprises fiber optic cables in contact with said culture wells.
 38. Thedevice of claim 26, wherein the solid support structure comprises one ormore microfluidics chambers.
 39. The device of claim 38, wherein the twoor more microfluidics chambers are operable connected to two or moreinlet microchannels and one or more outlet microchannels.
 40. A methodof preparing a device suitable for measuring a contractile function, themethod comprising providing a solid support structure; coating asacrificial polymer layer on the solid support structure; coating aflexible polymer layer that is more flexible than the support structureon the sacrificial polymer layer, wherein the flexible polymer layerdoes not cover the edges of said solid support structure; seeding cellson the flexible polymer layer; culturing the cells to form a tissue; andremoving a portion of said formed tissue thereby generating strips ofsaid formed tissue adhered at one end to said solid support structure,thereby preparing a device suitable for measuring a contractilefunction.
 41. The method of claim 40, further comprising applying aprotective film to one or more portions of the solid support structureprior to coating the solid support structure with the sacrificialpolymer layer.
 42. The method of claim 41, wherein one or more sectionsof the protective film are removed.
 43. The method of claim 40, whereina multi-well plate skeleton is attached to the rigid base material priorto seeding cells on the flexible polymer layer.
 44. The method of claim40, further comprising dividing the rigid base material into sectionsprior to seeding cells on the flexible polymer layer.
 45. The method ofclaim 44, wherein a of the rigid base material is fabricated into amicrochamber of a microfluidics device.
 46. The method of claim 44,wherein a section of the device is seeded with a first population ofcells, and a second section of the device is seeded with a secondpopulation of cells.
 47. The method of claim 44, wherein two or more ofthe individual sections of the device are combined following formationof the tissue.
 48. A method for measuring a contractile function,comprising: providing the device of claim 22; contacting the pluralityof said muscle thin films with a test compound; and determining theeffect of the test compound on a contractile function in the presenceand absence of the test compound, wherein a modulation of thecontractile function in the presence of said test compound as comparedto the contractile function in the absence of said test compoundindicates that said test compound modulates a contractile function,thereby identifying a compound that modulates a contractile function.49. The method of claim 48, further comprising applying a stimulus tocause the plurality of muscle thin films to move; and measuring theextend of movement of the plurality of muscle thin films.
 50. The methodof claim 48, wherein a radius of curvature of the plurality of musclethin films is measured.
 51. The method of claim 48, wherein a rate ofcontraction of the plurality of muscle thin films is measured.